P97 fragments with transfer activity

ABSTRACT

The present invention is related to fragments of human melanotransferrin (p97). In particular, this invention relates to treatment of diseases through the introduction of the melanotransferrin fragment conjugated to a therapeutic or diagnostic agent to a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/515,792, filed Aug. 5, 2011, which isincorporated by reference in its entirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BIOA_(—)004_(—)01US_ST25.txt. The text file isabout 32 KB, was created on Aug. 3, 2012, and is being submittedelectronically via EFS-Web.

BACKGROUND

1. Technical Field

The present invention is related to fragments of human melanotransferrin(p97). In particular, this invention relates to treatment of diseasesthrough the introduction of the melanotransferrin fragment conjugated toa therapeutic or diagnostic agent to a subject.

2. Description of the Related Art

Melanotransferrin (MTf) is a bi-lobed protein belonging to thetransferrin (Tf) family of iron binding proteins. It has beendemonstrated previously that MTf is able to directly bind and transportiron into mammalian cells independent of Tf and Tf receptor (TfR).Unlike other Tf family members, this molecule exists in two forms inhumans, a glycosyl-phosphatidylinositol (GPI)-linked cell surface formand a secreted water-soluble form. Additionally, MTf is also found to beexpressed on human brain endothelium where it is hypothesized totransport iron across the blood brain barrier (BBB). The role of MTf inthe transfer of iron into the brain was assessed by following bothradiolabeled soluble MTf and Tf into the mouse brain during a 24-hourperiod (Moroo et al., 2003, Demeule et al., 2002). It was determinedthat soluble MTf does have the ability to transcytose across theblood-brain barrier (BBB) and this transport was more efficient thanthat of Tf.

Subsequently, it has been demonstrated that soluble MTf could be used asa delivery vehicle of therapeutics into the brain (Karkan et al., 2008).Pharmacokinetics studies on soluble MTf demonstrated that the clearanceof MTf from serum was much greater than IgG control, and was rapidlydistributed to the tissues relative to IgG control. The transport ofsoluble MTf into the brain as a percentage of injected dose wassignificantly greater than IgG during the first hour post injection. Theaccumulation of soluble MTf in the brain was found to be significantlymore than that of IgG during the first 6-hours post injection.

Furthermore, it was shown that soluble MTf is able to deliver ironacross the BBB (Moroo et al., 2003), as well as paclitaxel covalentlylinked to MTf (Karkan et al., 2008). In the same study, while bothfree-adriamycin and MTf-adriamycin conjugates were able to equallyinhibit the subcutaneous growth of gliomas outside of the brain, onlyMTf-adriamycin conjugates significantly prolonged the survival ofanimals bearing intracranial gliomas when compared to thefree-adriamycin control (Karkan et al., 2008). Taken together, thesedata suggest soluble MTf as a potential drug delivery tool.

However, an even more efficient transfer molecule for delivering atarget agent would be useful for therapeutic and diagnostic purposes.The present invention addresses these and other needs.

BRIEF SUMMARY

Embodiments of the present invention include isolated p97(melanotransferrin; MTf) polypeptides consisting of the amino acidsequence set forth in SEQ ID NO:1-8 or 9. Also included are compositionscomprising a fragment of p97 consisting essentially of SEQ ID NO:1-8 or9 and a therapeutic or diagnostic agent.

In some embodiments, the p97 polypeptide is labeled with a labelselected from the group consisting of fluorescent molecules, luminescentmolecules, enzymes, substances having therapeutic activity, toxins, andradionuclides. In certain embodiments, the p97 polypeptide is conjugatedto a therapeutic agent or drug.

Particular embodiments include pharmaceutical compositions comprising atherapeutically effective amount of compound comprising a p97 fragmentcovalently linked to a therapeutic agent and a pharmaceuticallyacceptable excipient, wherein the p97 fragment consists of the aminoacid sequence set forth in SEQ ID NO:1-8 or 9.

Also included are compositions for delivering an agent across the bloodbrain barrier comprising a p97 fragment conjugated to the agent, asubstance which is capable of specifically binding to p97 conjugated tothe agent, or a p97 fragment fusion protein containing the p97 fragmentfused to the agent, and a pharmaceutically acceptable carrier ordiluent, wherein the p97 fragment consists of the amino acid sequenceset forth in SEQ ID NO:1-8 or 9.

Also included are conjugates, comprising a p97 polypeptide that consistsor consists essentially of SEQ ID NO:1-8 or 9, where the p97 polypeptideis covalently or operatively linked to an agent, to form a p97-agentconjugate. In some embodiments, the agent is a small molecule, apolypeptide, or a label (i.e., a detectable entity).

In particular embodiments, the small molecule is a cytotoxic orchemotherapeutic or anti-angiogenic agent selected from one or more ofalkylating agents, anti-metabolites, anthracyclines, anti-tumorantiobiotics, platinums, type I topoisomerase inhibitors, type IItopoisomerase inhibitors, vinca alkaloids, and taxanes. In specificembodiments, the small molecule is selected from one or more ofchlorambucil, cyclophosphamide, cilengitide, lomustine (CCNU),melphalan, procarbazine, thiotepa, carmustine (BCNU), enzastaurin,busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin,temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin,carboplatin, oxaliplatin, camptothecins, irinotecan, topotecan,amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus,everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923,paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitnib,vatalanib, geftinib, erlotinib, AEE-788, dichoroacetate, tamoxifen,fasudil, SB-681323, semaxanib, donepizil, galantamine, memantine,rivastigmine, tacrine, rasigiline, naltrexone, lubiprostone, safinamide,istradefylline, pimavanserin, pitolisant, isradipine, pridopidine(ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod, andmitoxantrone, including pharmaceutically acceptable salts and acidsthereof.

In some embodiments, the polypeptide is an antibody or antigen-bindingfragment thereof. In particular embodiments, the antibody orantigen-binding fragment thereof specifically binds to one or more ofhuman Her2/neu, Her1/EGFR, CD20, VEGF, CD52, CD33, CTLA-4, tenascin,alpha-4 (α4) integrin, IL-23, amyloid-β, Huntingtin, CD25, nerve growthfactor (NGF), TrkA, TNF-α, TNF-β, or α-synuclein, among other targetsdescribed herein.

In certain embodiments, the antibody is selected from one or more oftrastuzumab, cetuximab, daclizumab, tanezumab, 3F8, abagovomab,adalimumab, adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol),amatuximab, apolizumab, bavituximab, bectumomab, belimumab, bevacizumab,bivatuzumab (mertansine), brentuximab vedotin, cantuzumab (mertansine),cantuzumab (ravtansine), capromab (pendetide), catumaxomab,certolizumab, citatuzumab (bogatox), cixutumumab, clivatuzumab(tetraxetan), conatumumab, dacetuzumab, dalotuzumab, detumomab,drozitumab, ecromeximab, edrecolomab, elotuzumab, enavatuzumab,ensituximab, epratuzumab, ertumaxomab, etanercept, etaracizumab,farletuzumab, FBTA05, figitumumab, flanvotumab, galiximab, gemtuzumab,ganitumab, gemtuzumab (ozogamicin), girentuximab, glembatumumab(vedotin), golimumab, ibritumomab tiuxetan, icrucumab, igovomab,indatuximab ravtansine, infliximab, intetumumab, inotuzumab ozogamicin,ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab,lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab,matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab(pasudotox), nacolomab (tafenatox), naptumomab (estafenatox),narnatumab, necitumumab, nimotuzumab, nivolumab, Neuradiab® (with orwithout radioactive iodine), NR-LU-10, ofatumumab, olaratumab,onartuzumab, oportuzumab (monatox), oregovomab, panitumumab, patritumab,pemtumomab, pertuzumab, pritumumab, racotumomab, radretumab,ramucirumab, rilotumumab, rituximab, robatumumab, samalizumab,sibrotuzumab, siltuximab, tabalumab, taplitumomab (paptox), tenatumomab,teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650,tositumomab, TRBS07, tucotuzumab (celmoleukin), ublituximab, urelumab,veltuzumab, volociximab, votumumab, and zalutumumab, among otherantibodies described herein, and including antigen-binding fragmentsthereof.

In some embodiments, the polypeptide is an interferon-β polypeptide, oran active fragment or variant thereof.

In further embodiments, the polypeptide associates with a lysosomalstorage disease. In some aspects, the polypeptide is selected from oneor more of aspartylglucosaminidase, acid lipase, cysteine transporter,Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,β-hexosaminidase A, GM2-ganglioside activator (GM2A), α-D-mannosidase,β-D-mannosidase, arylsulfatase A, saposin B, neuraminidase,α-N-acetylglucosaminidase phosphotransferase, phosphotransferaseγ-subunit, L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase,α-N-acetylglucosaminidase, acetylCoA:N-acetyltransferase,N-acetylglucosamine 6-sulfatase, galactose 6-sulfatase, β-galactosidase,N-acetylgalactosamine 4-sulfatase, hyaluronoglucosaminidase, sulfatases,palmitoyl protein thioesterase, tripeptidyl peptidase I, acidsphingomyelinase, cathepsin A, cathepsin K, α-galactosidase B, NPC1,NPC2, sialin, and sialic acid transporter, including active fragmentsand variants thereof.

In particular embodiments, the detectable entity is selected from one ormore of diatrizoic acid, a radioisotope, a fluorophore/fluorescent dye,and a nanoparticle.

In some embodiments, the agent is a cardiotoxic agent in itsunconjugated form. Particular examples include where the cardiotoxicagent is an anthracycline/anthraquinolone, cyclophosphamide,antimetabolite, antimicrotubule agent, tyrosine kinase inhibitor,bevacizumab, or trastuzumab. Additional examples include where thecardiotoxic agent is cyclopentenyl cytosine, 5-fluorouracil,capecitabine, paclitaxel, docataxel, adriamycin, doxorubucin,epirubicin, emetine, isotamide, mitomycin C, erlotinib, gefitinib,imatinib, sorafenib, sunitinib, cisplatin, thalidomide, busulfan,vinblastine, bleomycin, vincristine, arsenic trioxide, methotrexate,rosiglitazone, or mitoxantrone.

Some embodiments include compositions (for example, pharmaceuticalcompositions), comprising a conjugate described herein, and apharmaceutically acceptable carrier.

Also included are methods of treating a subject in need thereof,comprising administering to the subject a conjugate or compositiondescribed herein.

Some methods are for treating a cancer of the central nervous system(CNS), optionally the brain. Particular methods are for treating primarycancer of the CNS, optionally the brain. Specific methods are fortreating a metastatic cancer of the CNS, optionally the brain. In someembodiments, the methods are for treating a glioma, meningioma,pituitary adenoma, vestibular schwannoma, primary CNS lymphoma,neuroblastoma, or primitive neuroectodermal tumor (medulloblastoma). Incertain aspects, the glioma is an astrocytoma, oligodendroglioma,ependymoma, or a choroid plexus papilloma.

Particular embodiments are for treating glioblastoma multiforme. Inspecific aspects, the glioblastoma multiforme is a giant cellgliobastoma or a gliosarcoma.

Certain methods are for treating a lysosomal storage disease. Exemplarylysosomal storage diseases include those selected from one or more ofaspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, Gaucher disease, globoid cellleucodystrophy, Krabbe disease, glycogen storage disease II, Pompedisease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I,Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, β-mannosidosis,metachromatic leucodystrophy, mucolipidosis type I, sialidosis typesI/II mucolipidosis types II/III I-cell disease, mucolipidosis type IIICpseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosistype IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII,Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatasedeficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease,Niemann-Pick disease types NB, Niemann-Pick disease, Niemann-Pickdisease type C1, Niemann-Pick disease type C2, pycnodysostosis,Schindler disease types I/II, Schindler disease, and sialic acid storagedisease.

Certain methods are for treating a degenerative or autoimmune disorderof the central nervous system (CNS). In some embodiments, thedegenerative or autoimmune disorder of the CNS is Alzheimer's disease,Huntington's disease, Parkinson's disease, or multiple sclerosis (MS).

In some embodiments, the subject is undergoing therapy with an otherwisecardiotoxic agent. Exemplary cardiotoxic agents includeanthracyclines/anthraquinolones, cyclophosphamides, antimetabolites,antimicrotubule agents, tyrosine kinase inhibitors, bevacizumab, andtrastuzumab. In some aspects, the cardiotoxic agent is cyclopentenylcytosine, 5-fluorouracil, capecitabine, paclitaxel, docataxel,adriamycin, doxorubucin, epirubicin, emetine, isotamide, mitomycin C,erlotinib, gefitinib, imatinib, sorafenib, sunitinib, cisplatin,thalidomide, busulfan, vinblastine, bleomycin, vincristine, arsenictrioxide, methotrexate, rosiglitazone, or mitoxantrone.

In some of the methods provided herein, the subject has cancer. Inparticular embodiments, the cancer is one or more of breast cancer,prostate cancer, gastrointestinal cancer, lung cancer, ovarian cancer,testicular cancer, head and neck cancer, stomach cancer, bladder cancer,pancreatic cancer, liver cancer, kidney cancer, squamous cell carcinoma,CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer,endometrial cancer, an epithelial tumor, bone cancer, or a hematopoieticcancer.

In some embodiments, administration of the conjugate reducescardiotoxicity of the agent, relative to an unconjugated form of theagent.

In certain aspects, the methods are for treating pain. In someembodiments, the pain is acute pain, chronic pain, neuropathic pain,and/or central pain. In particular embodiments, the pain is nociceptivepain, optionally visceral, deep somatic, or superficial somatic pain. Insome embodiments, the pain is breakthrough pain, and where the subjectis taking pain medication, and is optionally a subject with cancer pain.In further embodiments, the pain is incident pain. In certainembodiments, the pain has a central nervous system (CNS) component. Inparticular embodiments, the pain is osteoarthritis, chronic low backpain, bone cancer pain, or interstitial cystitis. In specificembodiments, the osteoarthritis is osteoarthritis of the knee, orosteoarthritis of the hip.

Also included are methods for imaging an organ or tissue component in asubject, comprising (a) administering to the subject a human p97polypeptide fragment of SEQ ID NO:1-8 or 9, where the polypeptide isconjugated to a detectable entity, and (b) visualizing the detectableentity in the subject. In some embodiments, the organ or tissuecompartment comprises the central nervous system. In particularembodiments, where the organ or tissue compartment comprises the brain.In certain aspects, visualizing the detectable entity comprises one ormore of fluoroscopy, projectional radiography, X-ray CT-scanning,positron emission tomography (PET), single photon emission computedtomography (SPECT), or magnetic resonance imaging (MRI).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the protein sequence alignment of human soluble p97 (H; SEQID NO:12) and mouse soluble p97(M; SEQ ID NO:13). The lightly shadedregion represents the amino acid sequence of a human soluble p97fragment (SEQ ID NO:1; or residues 1-564 of SEQ ID NO:12).

FIG. 2 shows a Coomassie blue stained native PAGE gel. Human p97 wasdigested over 3 days at 42° C. Lane 1 is human p97, lanes 2 and 3 arehuman p97 (3 mg) digested with hydroxylamine, and lane 4 is human p97 (5mg) digested with hydroxylamine.

FIG. 3 is a blot that shows the iodinated human p97 fragment (60 kDa).

FIG. 4 is a line graph that shows the percentage of ¹²⁵I radiolabeledp97 fragment present in the serum following delivery into mice throughtail vein injection.

FIG. 5 is a line graph that shows the percentage of the injected dose ofp97 fragment normalized to body mass (% ID/g BM) present in the brainover time.

FIG. 6 is a line graph that shows the ratio between radioactive counts(CPM) in one gram of tissue relative to one microliter of serum (Vd) inthe brain over time.

FIG. 7 is a line graph that shows the percent of the injected dose ofp97 fragment in the brain over 24 hours.

FIG. 8 is a line graph that shows the ratio between p97 fragment presentin the brain relative to serum over 24 hours.

FIG. 9 is a line graph that shows the percent of the injected dose ofp97 fragment in the heart over 24 hours.

FIG. 10 is a line graph that shows the ratio between p97 fragmentpresent in the heart relative to serum over 24 hours.

FIG. 11 is a line graph that shows the percent of the injected dose ofp97 fragment in the liver over 24 hours.

FIG. 12 is a line graph that shows the ratio between p97 fragmentpresent in the liver relative to serum over 24 hours.

FIG. 13 is a line graph that shows the percent of the injected dose ofp97 fragment in the kidney over 24 hours.

FIG. 14 is a line graph that shows the ratio between p97 fragmentpresent in the kidney relative to serum over 24 hours.

FIG. 15 is a line graph that shows the percent of the injected dose ofp97 fragment in the lung over 24 hours.

FIG. 16 is a line graph that shows the ratio between p97 fragmentpresent in the lung relative to serum over 24 hours.

FIG. 17 is a line graph that shows the percent of the injected dose ofp97 fragment in the spleen over 24 hours.

FIG. 18 is a line graph that shows the ratio between p97 fragmentpresent in the spleen relative to serum over 24 hours.

DETAILED DESCRIPTION

The present disclosure is based, in pertinent part, on the surprisingdiscovery that a smaller versions of soluble human MTf are able toretain the ability of melanotransferrin (MTf; p97) to cross the bloodbrain barrier (BBB). In particular, the present invention relates to thefragments of human MTf set forth in SEQ ID NOS:1-9 (see also FIG. 1).Embodiments of the invention pertain to the use of the p97 fragment forthe diagnosis, assessment and treatment of diseases and disorders,including, e.g., conditions involving disturbances in iron metabolism,Alzheimer's disease, cancers, and lysosomal storage diseases, amongothers. In specific embodiments, the invention relates to the p97fragment conjugated to a therapeutic or diagnostic agent.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. By “consisting of” is meant including, andlimited to, whatever follows the phrase “consisting of.” Thus, thephrase “consisting of” indicates that the listed elements are requiredor mandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey materially affect the activity or action of the listed elements.For certain polypeptide sequences, the phrase “consisting essentiallyof” can refer to polypeptides of essentially the same length as therecited polypeptide sequence, including those that differ by theaddition or deletion of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10N-terminal and/or C-terminal residues.

The term “conjugate” is intended to refer to the entity formed as aresult of covalent or non-covalent attachment or linkage of an agent orother molecule, e.g., a biologically active molecule, to a p97polypeptide. One example of a conjugate polypeptide is a “fusionprotein” or “fusion polypeptide,” that is, a polypeptide that is createdthrough the joining of two or more coding sequences, which originallycoded for separate polypeptides; translation of the joined codingsequences results in a single, fusion polypeptide, typically withfunctional properties derived from each of the separate polypeptides.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., Nucleic Acids Research. 12, 387-395, 1984), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated peptide” or an “isolated polypeptide” and thelike, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

The term “linkage,” “linker,” “linker moiety,” or “L” is used herein torefer to a linker that can be used to separate a p97 polypeptidefragment from an agent of interest, or to separate a first agent fromanother agent, for instance where two or more agents are linked to forma p97 conjugate. The linker may be physiologically stable or may includea releasable linker such as an enzymatically degradable linker (e.g.,proteolytically cleavable linkers). In certain aspects, the linker maybe a peptide linker, for instance, as part of a p97 fusion protein. Insome aspects, the linker may be a non-peptide linker ornon-proteinaceous linker. In some aspects, the linker may be particle,such as a nanoparticle.

The terms “modulating” and “altering” include “increasing,” “enhancing”or “stimulating,” as well as “decreasing” or “reducing,” typically in astatistically significant or a physiologically significant amount ordegree relative to a control. An “increased,” “stimulated” or “enhanced”amount is typically a “statistically significant” amount, and mayinclude an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30 or more times (e.g., 500, 1000 times) (including all integers anddecimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.)the amount produced by no composition (e.g., the absence of polypeptideof conjugate of the invention) or a control composition, sample or testsubject. A “decreased” or “reduced” amount is typically a “statisticallysignificant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%decrease in the amount produced by no composition or a controlcomposition, including all integers in between. As one non-limitingexample, a control could compare the activity, such as the amount orrate of transport/delivery across the blood brain barrier, the rateand/or levels of distribution to central nervous system tissue, and/orthe C_(max) for plasma, central nervous system tissues, or any othersystemic or peripheral non-central nervous system tissues, of ap97-agent conjugate relative to the agent alone. Other examples ofcomparisons and “statistically significant” amounts are describedherein.

In certain embodiments, the “purity” of any given agent (e.g., a p97polypeptide, a conjugate) in a composition may be specifically defined.For instance, certain compositions may comprise an agent that is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% pure, including all decimals in between, as measured, for exampleand by no means limiting, by high pressure liquid chromatography (HPLC),a well-known form of column chromatography used frequently inbiochemistry and analytical chemistry to separate, identify, andquantify compounds.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues and to variants and syntheticanalogues of the same. Thus, these terms apply to amino acid polymers inwhich one or more amino acid residues are synthetic non-naturallyoccurring amino acids, such as a chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally-occurring aminoacid polymers. The polypeptides described herein are not limited to aspecific length of the product; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide, and suchterms may be used interchangeably herein unless specifically indicatedotherwise. The polypeptides described herein may also comprisepost-expression modifications, such as glycosylations, acetylations,phosphorylations and the like, as well as other modifications known inthe art, both naturally occurring and non-naturally occurring. Apolypeptide may be an entire protein, or a subsequence, fragment,variant, or derivative thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include, but are not limitedto: carboxylate ester, phosphate ester, anhydride, acetal, ketal,acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester,carbonate, and hydrazone, peptides and oligonucleotides.

A “releasable linker” includes, but is not limited to, a physiologicallycleavable linker and an enzymatically degradable linker. Thus, a“releasable linker” is a linker that may undergo either spontaneoushydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed,acid-catalyzed, base-catalyzed, and so forth) under physiologicalconditions. For example, a “releasable linker” can involve anelimination reaction that has a base abstraction of a proton, (e.g., anionizable hydrogen atom, Hα), as the driving force. For purposes herein,a “releasable linker” is synonymous with a “degradable linker.” An“enzymatically degradable linkage” includes a linkage, e.g., amino acidsequence, that is subject to degradation by one or more enzymes, e.g.,peptidases or proteases. In particular embodiments, a releasable linkerhas a half life at pH 7.4, 25° C., e.g., a physiological pH, human bodytemperature (e.g., in vivo), of about 30 minutes, about 1 hour, about 2hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about12 hours, about 18 hours, about 24 hours, about 36 hours, about 48hours, about 72 hours, or about 96 hours or less.

The term “reference sequence” refers generally to a nucleic acid codingsequence, or amino acid sequence, to which another sequence is beingcompared. All polypeptide and polynucleotide sequences described hereinare included as references sequences, including those described by nameand those described in the Sequence Listing.

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% sequence identity to any of the reference sequences describedherein (see, e.g., Sequence Listing), typically where the polypeptidevariant maintains at least one biological activity of the referencepolypeptide.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity.” A “reference sequence” is atleast 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., Nucl. AcidsRes. 25:3389, 1997. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

The term “solubility” refers to the property of a p97 polypeptidefragment or conjugate to dissolve in a liquid solvent and form ahomogeneous solution. Solubility is typically expressed as aconcentration, either by mass of solute per unit volume of solvent (g ofsolute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity,molality, mole fraction or other similar descriptions of concentration.The maximum equilibrium amount of solute that can dissolve per amount ofsolvent is the solubility of that solute in that solvent under thespecified conditions, including temperature, pressure, pH, and thenature of the solvent. In certain embodiments, solubility is measured atphysiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0,or pH 7.4. In certain embodiments, solubility is measured in water or aphysiological buffer such as PBS or NaCl (with or without NaP). Inspecific embodiments, solubility is measured at relatively lower pH(e.g., pH 6.0) and relatively higher salt (e.g., 500 mM NaCl and 10 mMNaP). In certain embodiments, solubility is measured in a biologicalfluid (solvent) such as blood or serum. In certain embodiments, thetemperature can be about room temperature (e.g., about 20, 21, 22, 23,24, 25° C.) or about body temperature (˜37° C.). In certain embodiments,a p97 polypeptide or conjugate has a solubility of at least about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at roomtemperature or at about 37° C.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treated ordiagnosed with a p97 conjugate of the invention. Suitable subjects(patients) include laboratory animals (such as mouse, rat, rabbit, orguinea pig), farm animals, and domestic animals or pets (such as a cator dog). Non-human primates and, preferably, human patients, areincluded.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

“Substantially free” refers to the nearly complete or complete absenceof a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%,1%, 0.5% or less of some given quantity. For example, certaincompositions may be “substantially free” of cell proteins, membranes,nucleic acids, endotoxins, or other contaminants.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition, and may includeeven minimal changes or improvements in one or more measurable markersof the disease or condition being treated. “Treatment” or “treating”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof. The subjectreceiving this treatment is any subject in need thereof. Exemplarymarkers of clinical improvement will be apparent to persons skilled inthe art.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally-occurring source. A wild type gene or gene product (e.g., apolypeptide) is that which is most frequently observed in a populationand is thus arbitrarily designed the “normal” or “wild-type” form of thegene.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

Compositions and Preparation Thereof

In general, p97 fragment-conjugates may be prepared using techniqueswell known in the art. There are numerous approaches for the conjugationor chemical crosslinking of agents to a polypeptide such as the p97fragment, and one skilled in the art can determine which method is mostappropriate for conjugating a particular agent. The method employed mustbe capable of joining the agent with the p97 fragment withoutinterfering with the ability of the p97 fragment to bind to itsreceptor, preferably without influencing the biodistribution of the p97fragment-agent compared to the p97 fragment alone, and/or withoutsignificantly altering the desired activity of the agent (be ittherapeutic or prophylactic or the like) once delivered. A particularlypreferred method for linking complex molecules to the p97 fragment isthe SATA/sulfo-SMCC cross-linking reaction (Pierce (Rockford, Ill.)).

Methods of cross linking proteins and peptides are well known to thoseof skill in the art. Several hundred crosslinkers are available forconjugating a compound of interest with the p97 fragment or with asubstance which binds the p97 fragment (see, e.g., Chemistry of ProteinConjugation and Crosslinking, Shans Wong, CRC Press, Ann Arbor (1991)and U.S. Pat. No. 5,981,194 and PCT Patent Publication Nos. WO 02/13843and WO 01/59459 which are incorporated herein by reference in theirentirety). Many reagents and cross-linkers can be used to prepareconjugates of an active agent and a p97 fragment molecule. See, forinstance, Hermanson, G T et al. Bioconjugate Techniques, Academic Press,(1996). The crosslinker is generally chosen based on the reactivefunctional groups available or inserted on the therapeutic agent. Inaddition, if there are no reactive groups, a photoactivatiblecrosslinker can be used. In certain instances, it may be desirable toinclude a spacer between the p97 fragment and the agent. In oneembodiment, the p97 fragment and the protein therapeutic agents may beconjugated by the introduction of a sulfhydryl group on the p97 fragmentand by the introduction of a crosslinker containing a reactive thiolgroup on to the protein compound through carboxyl groups (Wawizynczakand Thorpe in Immunoconjugates: Antibody Conjugates in Radioimaging andTherapy of Cancer, Vogel (Ed.) Oxford University Press, pp. 28-55(1987); and Blair and Ghose (1983) J. Immunol. Methods 59:129). In someembodiments, the linker is vulnerable to hydrolysis at the acidic pH ofthe lysosome so as to free the agent from the p97 fragment and/orlinker.

In some embodiments of the present invention, the p97 fragment-agentconjugate is a p97 fragment-fusion protein. Fusion proteins may beprepared using standard techniques known in the art. Typically, a DNAmolecule encoding the p97 fragment or a portion thereof is linked to aDNA molecule encoding the protein compound. The chimeric DNA construct,along with suitable regulatory elements can be cloned into an expressionvector and expressed in a suitable host. The resultant fusion proteinscontain the p97 fragment fused to the selected protein compound.

When a linker is used, the linker is preferably an organic moietyconstructed to contain an alkyl, aryl and/or amino acid backbone, andcontaining an amide, ether, ester, hydrazone, disulphide linkage or anycombination thereof. Linkages containing amino acid, ether and amidebound components are stable under conditions of physiological pH,normally 7.4 in serum. Preferred linkages are those containing esters orhydrazones that are stable at serum pH, but that hydrolyze to releasethe drug when exposed to lysosomal pH. Disulphide linkages are preferredbecause they are sensitive to reductive cleavage. In addition, aminoacid linkers may be designed to be sensitive to cleavage by specificenzymes in the desired target organ or more preferably, the lysosomeitself. Exemplary linkers are described in Blattler et al. (19S5)Biochem. 24:1517-1524; King et al (1986) Biochem. 25:5774-5779;Srinivasachar and Nevill (1989) Biochem. 28:2501-2509.

In some embodiments, the linker is a polyethylene glycol orpolypropylene glycol. In other embodiments, the linker is from 4 to 20atoms long. In other embodiments, the linker is from 1 to 30 atoms longwith carbon chain atoms which may be substituted by heteroatomsindependently selected from the group consisting of O, N, or S. In someembodiments, from 1-4 or from 5 to 15 of the C atoms are substitutedwith a heteroatom independently selected from O, N, S. In otherembodiments, the linker contains a moiety subject to hydrolysis upondelivery to the lysosomal environment (e.g., susceptible to hydrolysisat the lysosomal pH or upon contact to a lysosomal enzyme). In someembodiments, the linker group is preferably hydrophilic to enhance thesolubility of the conjugate in body fluids. In some embodiments, thelinker contains or is attached to the p97 fragment molecule or theprotein agent by a functional group subject to attack by other lysosomalenzymes (e.g., enzymes not deficient in the target lysosome or alysosomal enzyme not conjugated to the p97 fragment carrier). In someembodiments, the p97 fragment and agent are joined by a linkercomprising amino acids or peptides. lipids, or sugar residues. In someembodiments, the p97 fragment and agent are joined at groups introducedsynthetically or by posttranslational modifications.

In some embodiments, agent-linker intermediates are similar to what hasbeen described previously, but comprise, for example, either an activeester that can react with free amine groups on the p97 fragment or amaleimide that can react with the free thiols created on the p97fragment via a SATA reaction or through other groups where personsskilled in the art can attach them to the p97 fragment.

p97 Sequences.

In some embodiments, a p97 polypeptide comprises, consists essentiallyof, or consists of at least one of the human p97 fragments identified inSEQ ID NO:1-8 or 9.

In other specific embodiments, a p97 polypeptide sequence comprises asequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identity or homology, along its length, to at least one of the humanp97 fragments identified in SEQ ID NO:1-8 or 9.

In particular embodiments, the p97 fragment or variant thereof has theability to cross the BBB, and optionally transport an agent of interestacross the BBB and into the central nervous system. In certainembodiments, the p97 fragment or variant thereof is capable ofspecifically binding to a p97 receptor, an LRP1 receptor, and/or an LRP1B receptor.

Preparation of p97

The p97 fragment for use in the methods and compositions of the presentinvention may be obtained, isolated or prepared from a variety ofsources.

In one aspect, standard recombinant DNA techniques may be used toprepare the p97 fragment. Within one embodiment, DNA encoding the p97fragment may be obtained by polymerase chain reaction (PCR)amplification of the p97 fragment sequence set forth in SEQ ID NO:1-8 or9 (see, generally, U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,800,159;see, also, PCR Technology: Principles and Applications for DNAAmplification, Erlich (ed.), Stockton Press (1989)). Briefly,double-stranded DNA from cells which express the p97 fragment (e.g.,SK-MEL-28 cells) is denatured by heating in the presence of heat stableTaq polymerase, sequence specific DNA primers such as 5′ GCGGACTTCCTCGG3′ (SEQ ID NO:10) and 5′ TCGCGAGCTTCCT 3′ (SEQ ID NO:11), ATP, CTP, GTPand TTP. Double-stranded DNA is produced when the synthesis is complete.This cycle may be repeated many times, resulting in a factorialamplification of the p97 fragment DNA. The amplified the p97 fragmentDNA may then be readily inserted into an expression vector as describedbelow.

Alternatively, DNA encoding the p97 fragment may be isolated using thecloning techniques described by Brown et al. in the UK PatentApplication No. GB 2188 637.

As noted above, the present invention provides recombinant expressionvectors which include either synthetic, or cDNA-derived DNA fragmentsencoding the p97 fragment, which are operably linked to suitabletranscriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, including,but not limited to, bacterial, fungal, viral, mammalian, and insectgenes. Selection of appropriate regulatory elements is dependent on thehost cell chosen, and may be readily accomplished by one of ordinaryskill in the art. Examples of regulatory elements include, inparticular, a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements, such as an origin ofreplication, additional DNA restriction sites, enhancers, sequencesconferring inducible transcription, and selectable markers, may beincorporated into the expression vector.

DNA sequences encoding the p97 fragment may be expressed by a widevariety of prokaryotic and eukaryotic host cells, including, but notlimited to, bacterial, mammalian, yeast, fungi, viral, plant, and insectcells. Methods for transforming or transfecting such cells forexpressing foreign DNA are well known in the art (see, e.g., Itakura etal, U.S. Pat. No. 4,704,362; Hinnen et al. (1978) PNAS USA 75:1929-1933;Murray et al, U.S. Pat. No. 4,801,542; Upshall et al, U.S. Pat. No.4,935,349; Hagen et al, U.S. Pat. No. 4,784,950; Axel et al, U.S. Pat.No. 4,399,216; Goeddel et al, U.S. Pat. No. 4,766,075; and Sambrook etal, supra).

Promoters, terminators, and methods for introducing expression vectorsof an appropriate type into, for example, plant, avian, and insect cellsmay be readily accomplished by those of skill in the art. Recombinantlyproduced p97 fragment may be further purified as described in moredetail below.

The soluble form of p97 may be prepared by culturing cells containingthe soluble p97 through the log phase of the cell's growth andcollecting the supernatant. Preferably, the supernatant is collectedprior to the time at which the cells lose viability. Soluble p97 maythen be purified as described below, in order to yield isolated solublep97. Suitable methods for purifying the soluble p97 can be selectedbased on the hydrophilic property of the soluble p97. For example, thesoluble p97 may be readily obtained by Triton X-I 14 Phase Separation.Once the soluble p97 has been purified, it may be digested with, e.g.,hydroxylamine as described in the Examples to generate the p97 fragment.

Therapeutic Agents

As noted above, certain embodiments comprise a p97 polypeptide that islinked to a therapeutic agent or drug of interest, for instance, a smallmolecule or a polypeptide (e.g., peptide, antibody). Also included areconjugates that comprise more than one therapeutic agent of interest,for instance, a p97 fragment conjugated to an antibody and a smallmolecule.

Covalent linkages are preferred, however, non-covalent linkages can alsobe employed, including those that utilize relatively strong non-covalentprotein-ligand interactions, such as the interaction between biotin andavidin. Operative linkages are also included, which do not necessarilyrequire a directly covalent or non-covalent interaction between the p97fragment and the agent of interest; examples of such linkages includeliposome mixtures that comprise a p97 polypeptide and an agent ofinterest. Exemplary methods of generating protein conjugates aredescribed herein, and other methods are well-known in the art.

Exemplary small molecules include cytotoxic, chemotherapeutic, andanti-angiogenic agents, for instance, those that have been considereduseful in the treatment of various cancers, including cancers of thecentral nervous system and cancers that have metastasized to the centralnervous system. Particular classes of small molecules include, withoutlimitation, alkylating agents, anti-metabolites, anthracyclines,anti-tumor antiobiotics, platinums, type I topoisomerase inhibitors,type II topoisomerase inhibitors, vinca alkaloids, and taxanes.

Specific examples of small molecules include chlorambucil,cyclophosphamide, cilengitide, lomustine (CCNU), melphalan,procarbazine, thiotepa, carmustine (BCNU), enzastaurin, busulfan,daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin,temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin,carboplatin, oxaliplatin, camptothecins, irinotecan, topotecan,amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus,everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923,and paclitaxel, and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Additional examples of small molecules include those that target proteinkinases for the treatment of nervous system (e.g., CNS) disorders,including imatinib, dasatinib, sorafenib, pazopanib, sunitnib,vatalanib, geftinib, erlotinib, AEE-788, dichoroacetate, tamoxifen,fasudil, SB-681323, and semaxanib (SU5416) (see Chico et al., Nat RevDrug Discov. 8:829-909, 2009). Examples of small molecules also includedonepizil, galantamine, memantine, rivastigmine, tacrine, rasigiline,naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin,pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, andbexarotene (e.g., for treating Alzheimer's Disease, Parkinson's Disease,Huntington's Disease); and glatirimer acetate, fingolimod, mitoxantrone(e.g., for treating MS). Also included are pharmaceutically acceptablesalts, acids or derivatives of any of the above.

Further examples of small molecules include alkylating agents such asthiotepa, cyclophosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (Taxol®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere®., Rhne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

Also included are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

As noted above, in certain aspects the small molecule is an otherwisecardiotoxic agent. Particular examples of cardiotoxic small moleculesinclude, without limitation, anthracyclines/anthraquinolones,cyclophosphamides, antimetabolites, antimicrotubule agents, and tyrosinekinase inhibitors. Specific examples of cardiotoxic agents includecyclopentenyl cytosine, 5-fluorouracil, capecitabine, paclitaxel,docataxel, adriamycin, doxorubucin, epirubicin, emetine, isotamide,mitomycin C, erlotinib, gefitinib, imatinib, sorafenib, sunitinib,cisplatin, thalidomide, busulfan, vinblastine, bleomycin, vincristine,arsenic trioxide, methotrexate, rosiglitazone, and mitoxantrone, amongother small molecules described herein and known in the art.

In particular embodiments, the therapeutic agent of interest is apeptide or polypeptide. The terms “peptide” and “polypeptide” are usedinterchangeably herein, however, in certain instances, the term“peptide” can refer to shorter polypeptides, for example, polypeptidesthat consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including allintegers and ranges (e.g., 5-10, 8-12, 10-15) in between. Polypeptidesand peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein. Antibodies arealso included as polypeptides.

Exemplary polypeptide agents include polypeptides associated withlysosomal storage disorders. Examples of such polypeptides includeaspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2,α-galactosidase A, acid ceramidase, α-L-fucosidase, β-hexosaminidase A,GM2-ganglioside activator (GM2A), α-D-mannosidase, β-D-mannosidase,arylsulfatase A, saposin B, neuraminidase, α-N-acetylglucosaminidasephosphotransferase, phosphotransferase γ-subunit, L-iduronidase,iduronate-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase,acetylCoA:N-acetyltransferase, N-acetylglucosamine 6-sulfatase,galactose 6-sulfatase, β-galactosidase, N-acetylgalactosamine4-sulfatase, hyaluronoglucosaminidase, sulfatases, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsinA, cathepsin K, α-galactosidase B, NPC1, NPC2, sialin, and sialic acidtransporter, including fragments, variants, and derivatives thereof.

Certain embodiments include polypeptides such as interferon-βpolypeptides, such as interferon-β1a (e.g., AVONEX, REBIF) andinterferon-β1b (e.g., Betaseron), which are often used for the treatmentof multiple sclerosis (MS).

In some embodiments, as noted above, the polypeptide agent is anantibody or an antigen-binding fragment thereof. The antibody orantigen-binding fragment used in the conjugates or compositions of thepresent invention can be of essentially any type. Particular examplesinclude therapeutic and diagnostic antibodies. As is well known in theart, an antibody is an immunoglobulin molecule capable of specificbinding to a target, such as a carbohydrate, polynucleotide, lipid,polypeptide, etc., through at least one epitope recognition site,located in the variable region of the immunoglobulin molecule.

As used herein, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), synthetic variantsthereof, naturally occurring variants, fusion proteins comprising anantibody portion with an antigen-binding fragment of the requiredspecificity, humanized antibodies, chimeric antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chains that binds to the antigen of interest. In thisregard, an antigen-binding fragment of the herein described antibodiesmay comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence fromantibodies that bind to a therapeutic or diagnostic target.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope is a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl, and may in certainembodiments have specific three-dimensional structural characteristics,and/or specific charge characteristics. Epitopes can be contiguous ornon-contiguous in relation to the primary structure of the antigen.

A molecule such as an antibody is said to exhibit “specific binding” or“preferential binding” if it reacts or associates more frequently, morerapidly, with greater duration and/or with greater affinity with aparticular cell or substance than it does with alternative cells orsubstances. An antibody “specifically binds” or “preferentially binds”to a target if it binds with greater affinity, avidity, more readily,and/or with greater duration than it binds to other substances. Forexample, an antibody that specifically or preferentially binds to aspecific epitope is an antibody that binds that specific epitope withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other epitopes. It is also understood by reading thisdefinition that, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Weals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(d) represents a greater affinity.Immunological binding properties of selected polypeptides can bequantified using methods well known in the art. One such method entailsmeasuring the rates of antigen-binding site/antigen complex formationand dissociation, wherein those rates depend on the concentrations ofthe complex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (K_(on)) and the “off rate constant”(K_(off)) can be determined by calculation of the concentrations and theactual rates of association and dissociation. The ratio ofK_(off)/K_(on) enables cancellation of all parameters not related toaffinity, and is thus equal to the dissociation constant K_(d).

Immunological binding properties of selected antibodies and polypeptidescan be quantified using methods well known in the art (see Davies etal., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, anantibody or other polypeptide is said to specifically bind an antigen orepitope thereof when the equilibrium dissociation constant is about≦10⁻⁷ or 10⁻⁸ M. In some embodiments, the equilibrium dissociationconstant of an antibody may be about ≦10⁻⁹ M or ≦10⁻¹⁰ M. In certainillustrative embodiments, an antibody or other polypeptide has anaffinity (K_(d)) for an antigen or target described herein (to which itspecifically binds) of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or50 nM.

In some embodiments, the antibody or antigen-binding fragment or otherpolypeptide specifically binds to a cell surface receptor or other cellsurface protein. In some embodiments, the antibody or antigen-bindingfragment or other polypeptide specifically binds to a ligand of a cellsurface receptor or other cell surface protein. In some embodiments, theantibody or antigen-binding fragment or other polypeptide specificallybinds to an intracellular protein.

In certain embodiments, the antibody or antigen-binding fragment thereofor other polypeptide specifically binds to a cancer-associated antigen,or cancer antigen. Exemplary cancer antigens include cell surfaceproteins such as cell surface receptors. Also included ascancer-associated antigens are ligands that bind to such cell surfaceproteins or receptors. In specific embodiments, the antibody orantigen-binding fragment specifically binds to a intracellular cancerantigen. In some embodiments, the cancer that associates with the cancerantigen is one or more of breast cancer, metastatic brain cancer,prostate cancer, gastrointestinal cancer, lung cancer, ovarian cancer,testicular cancer, head and neck cancer, stomach cancer, bladder cancer,pancreatic cancer, liver cancer, kidney cancer, squamous cell carcinoma,CNS or brain cancer, melanoma, non-melanoma cancer, thyroid cancer,endometrial cancer, epithelial tumor, bone cancer, or a hematopoieticcancer.

In particular embodiments, the antibody or antigen-binding fragment orother polypeptide specifically binds to at least one cancer-associatedantigen, or cancer antigen, such as human Her2/neu, Her1/EGF receptor(EGFR), Her3, A33 antigen, CD5, CD19, CD20, CD22, CD23 (IgE Receptor),C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF(e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51,CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4,NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor(IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1),carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), integrinα_(v)β₃, integrin α₅β₁, folate receptor 1, transmembrane glycoproteinNMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72,MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membraneantigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factorreceptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM familymember 7 (SLAMF7), EGP40 pancarcinoma antigen, B-cell activating factor(BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM(17-1A), Programmed Death-1, protein disulfide isomerase (PDI),Phosphatase of Regenerating Liver 3 (PRL-3), prostatic acid phosphatase,Lewis-Y antigen, GD2 (a disialoganglioside expressed on tumors ofneuroectodermal origin), glypican-3 (GPC3), and/or mesothelin.

In specific embodiments, the antibody or antigen-binding fragmentthereof or other polypeptide specifically binds to the human Her2/neuprotein. Essentially any anti-Her2/neu antibody, antigen-bindingfragment or other Her2/neu-specific binding agent may be used inproducing the p97-antibody conjugates of the present invention.Illustrative anti-Her2/neu antibodies are described, for example, inU.S. Pat. Nos. 5,677,171; 5,720,937; 5,720,954; 5,725,856; 5,770,195;5,772,997; 6,165,464; 6,387,371; and 6,399,063, the contents of whichare incorporated herein by reference in their entireties.

In some embodiments, the antibody or antigen-binding fragment thereof orother polypeptide specifically binds to the human Her1/EGFR (epidermalgrowth factor receptor). Essentially any anti-Her1/EGFR antibody,antigen-binding fragment or other Her1-EGFR-specific binding agent maybe used in producing the p97-antibody conjugates of the presentinvention. Illustrative anti-Her1/EGFR antibodies are described, forexample, in U.S. Pat. Nos. 5,844,093; 7,132,511; 7,247,301; 7,595,378;7,723,484; 7,939,072; and 7,960,516, the contents of which areincorporated by reference in their entireties.

In certain embodiments, the antibody is a therapeutic antibody, such asan anti-cancer therapeutic antibody, including antibodies such as 3F8,abagovomab, adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol),amatuximab, apolizumab, bavituximab, bectumomab, belimumab, bevacizumab,bivatuzumab (mertansine), brentuximab vedotin, cantuzumab (mertansine),cantuzumab (ravtansine), capromab (pendetide), catumaxomab, cetuximab,citatuzumab (bogatox), cixutumumab, clivatuzumab (tetraxetan),conatumumab, dacetuzumab, dalotuzumab, detumomab, drozitumab,ecromeximab, edrecolomab, elotuzumab, enavatuzumab, ensituximab,epratuzumab, ertumaxomab, etaracizumab, farletuzumab, FBTA05,figitumumab, flanvotumab, galiximab, gemtuzumab, ganitumab, gemtuzumab(ozogamicin), girentuximab, glembatumumab (vedotin), ibritumomabtiuxetan, icrucumab, igovomab, indatuximab ravtansine, intetumumab,inotuzumab ozogamicin, ipilimumab (MDX-101), iratumumab, labetuzumab,lexatumumab, lintuzumab, lorvotuzumab (mertansine), lucatumumab,lumiliximab, mapatumumab, matuzumab, milatuzumab, mitumomab,mogamulizumab, moxetumomab (pasudotox), nacolomab (tafenatox),naptumomab (estafenatox), narnatumab, necitumumab, nimotuzumab,nivolumab, Neuradiab® (with or without radioactive iodine), NR-LU-10,ofatumumab, olaratumab, onartuzumab, oportuzumab (monatox), oregovomab,panitumumab, patritumab, pemtumomab, pertuzumab, pritumumab,racotumomab, radretumab, ramucirumab, rilotumumab, rituximab,robatumumab, samalizumab, sibrotuzumab, siltuximab, tabalumab,taplitumomab (paptox), tenatumomab, teprotumumab, TGN1412, ticilimumab,tremelimumab, tigatuzumab, TNX-650, tositumomab, TRBS07, trastuzumab,tucotuzumab (celmoleukin), ublituximab, urelumab, veltuzumab,volociximab, votumumab, and zalutumumab. Also included are fragments,variants, and derivatives of these antibodies.

In particular embodiments, the antibody is a cardiotoxic antibody, thatis, an antibody that displays cardiotoxicity when administered in anunconjugated form. Specific examples of antibodies that displaycardiotoxicity include trastuzumab and bevacizumab.

In specific embodiments, the anti-Her2/neu antibody used in a p97conjugate is trastuzumab (Herceptin®), or a fragment, variant orderivative thereof. Herceptin® is a Her2/neu-specific monoclonalantibody approved for the treatment of human breast cancer. In certainembodiments, a Her2/neu-binding antigen-binding fragment comprises oneor more of the CDRs of a Her2/neu antibody. In this regard, it has beenshown in some cases that the transfer of only the VHCDR3 of an antibodycan be performed while still retaining desired specific binding (Barbaset al., PNAS. 92: 2529-2533, 1995). See also, McLane et al., PNAS USA.92:5214-5218, 1995; and Barbas et al., J. Am. Chem. Soc. 116:2161-2162,1994.

In other specific embodiments, the anti-Her1/EGFR antibody used in aconjugate of the invention is cetuximab (Erbitux®), or a fragment orderivative thereof. In certain embodiments, an anti-Her1/EGFR bindingfragment comprises one or more of the CDRs of a Her1/EGFR antibody suchas cetuximab. Cetuximab is approved for the treatment of head and neckcancer, and colorectal cancer. Cetuximab is composed of the Fv(variable; antigen-binding) regions of the 225 murine EGFR monoclonalantibody specific for the N-terminal portion of human EGFR with humanIgG1 heavy and kappa light chain constant (framework) regions.

In some embodiments, the antibody or antigen-binding fragment or otherpolypeptide specifically binds to an antigen associated with (e.g.,treatment of) at least one nervous system disorder, including disordersof the peripheral and/or central nervous system (CNS) disorder. Incertain embodiments, the antibody or antigen-binding fragment or otherpolypeptide specifically binds to an antigen associated with (e.g.,treatment of) pain, including acute pain, chronic pain, and neuropathicpain. In some embodiments, the antibody or antigen-binding fragment orother polypeptide specifically binds an antigen associated with (e.g.,treatment of) an autoimmune disorder, including autoimmune disorders ofthe nervous system or CNS.

Examples of nervous system-, pain-, and/or autoimmune-associatedantigens include, without limitation, alpha-4 (α4) integrin, tumornecrosis factor (TNF), IL-12, IL-23, the p40 subunit of IL-12 and IL-23,CD20, CD52, amyloid-β (e.g., Aβ₍₁₋₄₂₎), Huntingtin, CD25 (i.e., thealpha chain of the IL-2 receptor), nerve growth factor (NGF),neurotrophic tyrosine kinase receptor type 1 (TrkA; the high affinitycatalytic receptor for NGF), and α-synuclein. These targets have beenconsidered useful in the treatment of a variety of nervous system, pain,and/or autoimmune disorders, such as multiple sclerosis (α4 integrin,IL-23, CD25, CD₂₀, CD52, IL-12, IL-23, the p40 subunit of IL-12 andIL-23, Nogo-A, LINGO-1), Alzheimer's Disease (Aβ, TNF), Huntington'sDisease (Huntingtin), Parkinson's Disease (α-synuclein), and pain (NGFand TrkA).

In specific embodiments, the anti-CD25 antibody used in a p97 conjugateis daclizumab (i.e., Zenapax™), or a fragment, variant or derivativethereof. Daclizumab a humanized monoclonal antibody that specificallybinds to CD25, the alpha subunit of the IL-2 receptor. In someembodiments, the antibody is natalizumab, or a variant or fragmentthereof that specifically binds to α4 integrin. In other embodiments,the antibody is rituximab, ocrelizumab, ofatumumab, or a variant orfragment thereof that specifically binds to CD₂₀. In particularembodiments, the antibody is alemtuzumab, or a variant or fragmentthereof that specifically binds to CD52. In certain embodiments, theantibody is ustekinumab (CNTO 1275), or a variant or fragment thereofthat specifically binds to the p40 subunit of IL-12 and IL-23.

In specific embodiments, the anti-NGF antibody used in a conjugate istanezumab, or a fragment, variant or derivative thereof. Tanezumabspecifically binds to NGF and prevents NGF from binding to its highaffinity, membrane-bound, catalytic receptor tropomyosin-related kinaseA (TrkA), which is present on sympathetic and sensory neurons; reducedstimulation of TrkA by NGF is believed to inhibit the pain-transmissionactivities of such neurons.

In some embodiments, the antibody used in a conjugate specifically bindsto tumor necrosis factor (TNF)-α or TNF-β. In specific embodiments, theanti-TNF antibody is adalimumab (Humira®), certolizumab pegol (Cimzia®),etanercept (Enbrel®), golimumab (Cimzia®), or infliximab (Remicade®),D2E7, CDP 571, or CDP 870, or an antigen-binding fragment or variantthereof. Conjugates comprising an anti-TNF antibody can be used, forinstance, in the treatment of neurological conditions or disorders suchas Alzheimer's disease, stroke, traumatic brain injury (TBI), spinalstenosis, acute spinal cord injury, spinal cord compression (see U.S.Pat. Nos. 6,015,557; 6,177,077; 6,419,934; 6,419,944; 6,537,549;6,982,089; and 7,214,658).

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the Velocimmune® platform by Regenerex®(see, e.g., U.S. Pat. No. 6,596,541).

Antibodies can also be generated or identified by the use of phagedisplay or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592;Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examplesof available libraries include cloned or synthetic libraries, such asthe Human Combinatorial Antibody Library (HuCAL), in which thestructural diversity of the human antibody repertoire is represented byseven heavy chain and seven light chain variable region genes. Thecombination of these genes gives rise to 49 frameworks in the masterlibrary. By superimposing highly variable genetic cassettes(CDRs=complementarity determining regions) on these frameworks, the vasthuman antibody repertoire can be reproduced. Also included are humanlibraries designed with human-donor-sourced fragments encoding alight-chain variable region, a heavy-chain CDR-3, synthetic DNA encodingdiversity in heavy-chain CDR-1, and synthetic DNA encoding diversity inheavy-chain CDR-2. Other libraries suitable for use will be apparent topersons skilled in the art. The p97 polypeptides described herein andknown in the art may be used in the purification process in, forexample, an affinity chromatography step.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific, being directed against a single epitope. The term “monoclonalantibody” encompasses not only intact monoclonal antibodies andfull-length monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusionproteins comprising an antigen-binding portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals). The term includes whole immunoglobulins as well asthe fragments etc. described above under the definition of “antibody.”

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments of the present invention can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions of an IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. See Inbar et al.,PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710,1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

In certain embodiments, single chain Fv or scFV antibodies arecontemplated. For example, Kappa bodies (Ill et al., Prot. Eng.10:949-57, 1997); minibodies (Martin et al., EMBO J. 13:5305-9, 1994);diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins(Traunecker et al., EMBO J. 10: 3655-59, 1991; and Traunecker et al.,Int. J. Cancer Suppl. 7:51-52, 1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity.

A single chain Fv (sFv) polypeptide is a covalently linked V_(H)::V_(L)heterodimer which is expressed from a gene fusion including V_(H)- andV_(L)-encoding genes linked by a peptide-encoding linker. Huston et al.(PNAS USA. 85(16):5879-5883, 1988). A number of methods have beendescribed to discern chemical structures for converting the naturallyaggregated—but chemically separated—light and heavy polypeptide chainsfrom an antibody V region into an sFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405,to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

In certain embodiments, an antibody as described herein is in the formof a “diabody.” Diabodies are multimers of polypeptides, eachpolypeptide comprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804). A dAb fragment of an antibody consists of a VH domain(Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalentor multispecific fragments can be constructed, for example, by genefusion (see WO94/13804; and Holliger et al., PNAS USA. 90:6444-6448,1993)).

Minibodies comprising a scFv joined to a CH3 domain are also included(see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al.,Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988;Huston et al., PNAS USA. 85:5879-5883, 1988); PCT/US92/09965;WO94/13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. Diabodies and scFv can beconstructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(Ridgeway et al., Protein Eng., 9:616-621, 1996).

In certain embodiments, the antibodies described herein may be providedin the form of a UniBody®. A UniBody® is an IgG4 antibody with the hingeregion removed (see GenMab Utrecht, The Netherlands; see also, e.g., USApplication No. 2009/0226421). This antibody technology creates astable, smaller antibody format with an anticipated longer therapeuticwindow than current small antibody formats. IgG4 antibodies areconsidered inert and thus do not interact with the immune system. Fullyhuman IgG4 antibodies may be modified by eliminating the hinge region ofthe antibody to obtain half-molecule fragments having distinct stabilityproperties relative to the corresponding intact IgG4 (GenMab, Utrecht).Halving the IgG4 molecule leaves only one area on the UniBody® that canbind to cognate antigens (e.g., disease targets) and the UniBody®therefore binds univalently to only one site on target cells. Forcertain cancer cell surface antigens, this univalent binding may notstimulate the cancer cells to grow as may be seen using bivalentantibodies having the same antigen specificity, and hence UniBody®technology may afford treatment options for some types of cancer thatmay be refractory to treatment with conventional antibodies. The smallsize of the UniBody® can be a great benefit when treating some forms ofcancer, allowing for better distribution of the molecule over largersolid tumors and potentially increasing efficacy.

In certain embodiments, the antibodies provided herein may take the formof a nanobody. Minibodies are encoded by single genes and areefficiently produced in almost all prokaryotic and eukaryotic hosts, forexample, E. coli (see U.S. Pat. No. 6,765,087), moulds (for exampleAspergillus or Trichoderma) and yeast (for example Saccharomyces,Kluyvermyces, Hansenula or Pichia (see U.S. Pat. No. 6,838,254). Theproduction process is scalable and multi-kilogram quantities ofnanobodies have been produced. Nanobodies may be formulated as aready-to-use solution having a long shelf life. The Nanoclone method(see WO 06/079372) is a proprietary method for generating Nanobodiesagainst a desired target, based on automated high-throughput selectionof B-cells.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof are humanized. These embodiments refer to a chimeric molecule,generally prepared using recombinant techniques, having anantigen-binding site derived from an immunoglobulin from a non-humanspecies and the remaining immunoglobulin structure of the molecule basedupon the structure and/or sequence of a human immunoglobulin. Theantigen-binding site may comprise either complete variable domains fusedonto constant domains or only the CDRs grafted onto appropriateframework regions in the variable domains. Epitope binding sites may bewild type or modified by one or more amino acid substitutions. Thiseliminates the constant region as an immunogen in human individuals, butthe possibility of an immune response to the foreign variable regionremains (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al.,PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327,1988). Illustrative methods for humanization of antibodies include themethods described in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann etal., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536,1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maedaet al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNASUSA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991;Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA.89:4285-4289, 1992; and Co et al., J. Immunol. 148:1149-1154, 1992. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies of the present invention may bechimeric antibodies. In this regard, a chimeric antibody is comprised ofan antigen-binding fragment of an antibody operably linked or otherwisefused to a heterologous Fc portion of a different antibody. In certainembodiments, the heterologous Fc domain is of human origin. In otherembodiments, the heterologous Fc domain may be from a different Ig classfrom the parent antibody, including IgA (including subclasses IgA1 andIgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4),and IgM. In further embodiments, the heterologous Fc domain may becomprised of CH2 and CH3 domains from one or more of the different Igclasses. As noted above with regard to humanized antibodies, theantigen-binding fragment of a chimeric antibody may comprise only one ormore of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4,5, or 6 CDRs of the antibodies described herein), or may comprise anentire variable domain (VL, VH or both).

Labels

In some embodiments, the p97 fragment conjugate is labeled to facilitateits detection. A “label” or a “detectable entity” is a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. For example, labels suitable for usein the present invention include, for example, radioactive labels (e.g.,³²P), fluorophores (e.g., fluorescein), electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins which can be made detectable, e.g., by incorporating aradiolabel into the hapten or peptide, or used to detect antibodiesspecifically reactive with the hapten or peptide.

As noted above, depending on the screening assay employed, the agent,the linker or the p97 fragment portion of a conjugate may be labeled.The particular label or detectable group used is not a critical aspectof the invention, as long as it does not significantly interfere withthe biological activity of the conjugate. The detectable group can beany material having a detectable physical or chemical property. Thus, alabel is any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.

Examples of labels suitable for use in the present invention include,but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,H, I, S, C, or P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. Preferably, thelabel in one embodiment is covalently bound to the p97 fragment using anisocyanate reagent for conjugating an active agent according to theinvention. In one aspect of the invention, the bifunctional isocyanatereagents of the invention can be used to conjugate a label to the p97fragment to form a label p97 fragment conjugate without an active agentattached thereto. The label p97 fragment conjugate may be used as anintermediate for the synthesis of a labeled conjugate according to theinvention or may be used to detect the p97 fragment conjugate. Asindicated above, a wide variety of labels can be used, with the choiceof label depending on sensitivity required, ease of conjugation with thedesired component of the assay, stability requirements, availableinstrumentation, and disposal provisions. Non-radioactive labels areoften attached by indirect means. Generally, a ligand molecule (e.g.,biotin) is covalently bound to the molecule. The ligand then binds toanother molecules (e.g., streptavidin) molecule, which is eitherinherently detectable or covalently bound to a signal system, such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound.

The conjugates can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymessuitable for use as labels include, but are not limited to, hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds, i.e., fluorophores,suitable for use as labels include, but are not limited to, fluoresceinand its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Further examples of suitable fluorophores include,but are not limited to, eosin, TRITC-amine, quinine, fluorescein W,acridine yellow, lissamine rhodamine, B sulfonyl chloride erythroscein,ruthenium (tris, bipyridinium), Texas Red, nicotinamide adeninedinucleotide, flavin adenine dinucleotide, etc. Chemiluminescentcompounds suitable for use as labels include, but are not limited to,luciferin and 2,3-dihydrophthalazinediones, e.g., luminol. For a reviewof various labeling or signal producing systems that can be used in themethods of the present invention, see U.S. Pat. No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Other labeling anddetection systems suitable for use in the methods of the presentinvention will be readily apparent to those of skill in the art. Suchlabeled modulators and ligands may be used in the diagnosis of a diseaseor health condition.

Pharmaceutical Compositions, and Methods of Use/Treatment/Administration

Certain embodiments of the present invention relate to methods of usingthe compositions of p97 polypeptides and p97 conjugates describedherein. Examples of such methods include methods of treatment andmethods of diagnosis, including for instance, the use of p97 conjugatesfor medical imaging of certain organs/tissues, such as those of thenervous system. Specific embodiments include methods of diagnosingand/or treating disorders or conditions of the central nervous system(CNS), or disorders or conditions having a CNS component.

Accordingly, certain embodiments include methods of treating a subjectin need thereof, comprising administering a composition that comprises ap97 conjugate described herein. Also included are methods of deliveringan agent to the nervous system (e.g., central nervous system tissues) ofa subject, comprising administering a composition that comprises a p97conjugate described herein. In certain of these and related embodiments,the methods increase the rate of delivery of the agent to the centralnervous system tissues, relative, for example, to delivery by acomposition that comprises the agent alone.

In some instances, a subject has a disease, disorder, or condition ofthe CNS, where increased delivery of a therapeutic agent across theblood brain barrier to CNS tissues relative to peripheral tissues canimprove treatment, for instance, by reducing side-effects associatedwith exposure of an agent to peripheral tissues. Exemplary diseases,disorders, and conditions of the CNS include various cancers, includingprimary and metastatic CNS cancers, lysosomal storage diseases,neurodegenerative diseases such as Alzheimer's disease, and auto-immunediseases such as multiple sclerosis.

Certain embodiments thus relate to methods for treating a cancer of thecentral nervous system (CNS), optionally the brain, where the subject inneed thereof has such a cancer or is at risk for developing such acondition. In some embodiments, the cancer is a primary cancer of theCNS, such as a primary cancer of the brain. For instance, the methodscan be for treating a glioma, meningioma, pituitary adenoma, vestibularschwannoma, primary CNS lymphoma, or primitive neuroectodermal tumor(medulloblastoma). In some embodiments, the glioma is an astrocytoma,oligodendroglioma, ependymoma, or a choroid plexus papilloma. In certainembodiments, the primary CNS or brain cancer is glioblastoma multiforme,such as a giant cell gliobastoma or a gliosarcoma.

In particular embodiments, the cancer is a metastatic cancer of the CNS,for instance, a cancer that has metastasized to the brain. Examples ofsuch cancers include, without limitation, breast cancers, lung cancers,genitourinary tract cancers, gastrointestinal tract cancers (e.g.,colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas,head and neck cancers, prostate cancers (e.g., prostaticadenocarcinomas), and lymphomas. Certain embodiments thus includemethods for treating, inhibiting or preventing metastasis of a cancer byadministering to a patient a therapeutically effective amount of aherein disclosed conjugate (e.g., in an amount that, followingadministration, inhibits, prevents or delays metastasis of a cancer in astatistically significant manner, i.e., relative to an appropriatecontrol as will be known to those skilled in the art). In particularembodiments, the subject has a cancer that has not yet metastasized tothe central nervous system, including one or more of the above-describedcancers, among others known in the art.

In particular embodiments, the cancer (cell) expresses or overexpressesone or more of Her2/neu, CD₂₀, Her1/EGF receptor(s), VEGF receptor(s),PDGF receptor(s), CD30, CD52, CD33, CTLA-4, or tenascin.

Also included is the treatment of other cancers, including breastcancer, prostate cancer, gastrointestinal cancer, lung cancer, ovariancancer, testicular cancer, head and neck cancer, stomach cancer, bladdercancer, pancreatic cancer, liver cancer, kidney cancer, squamous cellcarcinoma, melanoma, non-melanoma cancer, thyroid cancer, endometrialcancer, epithelial tumor, bone cancer, or a hematopoietic cancer. Hence,in certain embodiments, the cancer cell being treated by a p97 conjugateoverexpresses or is associated with a cancer antigen, such as humanHer2/neu, Her1/EGF receptor (EGFR), Her3, A33 antigen, CD5, CD19, CD₂₀,CD22, CD23 (IgE Receptor), C242 antigen, 5T4, IL-6, IL-13, vascularendothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30,CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200,CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-likegrowth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-likegrowth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonicantigen (CEA), integrin α_(v)β₃, integrin α₅β₁, folate receptor 1,transmembrane glycoprotein NMB, fibroblast activation protein alpha(FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and/or mesothelin.

The use of p97 conjugates for treating cancers including cancers of theCNS can be combined with other therapeutic modalities. For example, acomposition comprising a p97 conjugate can be administered to a subjectbefore, during, or after other therapeutic interventions, includingsymptomatic care, radiotherapy, surgery, transplantation, immunotherapy,hormone therapy, photodynamic therapy, antibiotic therapy, or anycombination thereof. Symptomatic care includes administration ofcorticosteroids, to reduce cerebral edema, headaches, cognitivedysfunction, and emesis, and administration of anti-convulsants, toreduce seizures. Radiotherapy includes whole-brain irradiation,fractionated radiotherapy, and radiosurgery, such as stereotacticradiosurgery, which can be further combined with traditional surgery.

In specific combination therapies, the antibody portion of anp97-antibody conjugate comprises cetuximab, and the p97-cetuximabconjugate is used for treating a subject with locally or regionallyadvanced squamous cell carcinoma of the head and neck in combinationwith radiation therapy. In other aspects, the p97-cetuximab conjugate isused for treating a subject with recurrent locoregional disease ormetastatic squamous cell carcinoma of the head and neck in combinationwith platinum-based therapy with 5-fluorouracil (5-FU). In some aspects,the p97-cetuximab conjugate is used in combination with irinotecan fortreating a subject with EGFR-expressing colorectal cancer and that isrefractory to irinotecan-based chemotherapy.

In some instances, the subject has or is at risk for having a lysosomalstorage disease. Certain methods thus relate to the treatment oflysosomal storage diseases in a subject in need thereof, optionallythose lysosomal storage diseases associated with the central nervoussystem. Exemplary lysosomal storage diseases includeaspartylglucosaminuria, cholesterol ester storage disease, Wolmandisease, cystinosis, Danon disease, Fabry disease, Farberlipogranulomatosis, Farber disease, fucosidosis, galactosialidosis typesI/II, Gaucher disease types I/II/III, Gaucher disease, globoid cellleucodystrophy, Krabbe disease, glycogen storage disease II, Pompedisease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I,Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease,GM2-gangliosidosis, α-mannosidosis types I/II, β-mannosidosis,metachromatic leucodystrophy, mucolipidosis type I, sialidosis typesI/II mucolipidosis types II/III I-cell disease, mucolipidosis type IIICpseudo-Hurler polydystrophy, mucopolysaccharidosis type I,mucopolysaccharidosis type II, Hunter syndrome, mucopolysaccharidosistype IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB,mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID,mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosistype IVB Morquio syndrome, mucopolysaccharidosis type VI,mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis typeIX, multiple sulfatase deficiency, neuronal ceroid lipofuscinosis, CLN1Batten disease, Niemann-Pick disease types NB, Niemann-Pick disease,Niemann-Pick disease type C1, Niemann-Pick disease type C2,pycnodysostosis, Schindler disease types I/II, Schindler disease, andsialic acid storage disease. In these and related embodiments, the p97polypeptide can be conjugated to one or more polypeptides associatedwith a lysosomal storage disease, as described herein.

In certain instances, the subject has or is at risk for having anauto-immune disorder and/or a neurodegenerative or other neurologicaldisorder, optionally of the CNS. Hence, also included are methods oftreating a degenerative or autoimmune disorder of the central nervoussystem (CNS) in a subject in need thereof. For instance, in specificembodiments, the degenerative or autoimmune disorder of the CNS isAlzheimer's disease, Huntington's disease, Parkinson's disease, ormultiple sclerosis (MS). Hence, certain embodiments includeadministering a p97 conjugate to a subject having Alzheimer's disease,Huntington's disease, Parkinson's disease, or MS. In particularembodiments, the p97 polypeptide is conjugated to an antibody or otheragent that specifically binds to amyloid-β (e.g., Aβ₍₁₋₄₂₎) or tumornecrosis factor (TNF-α, TNF-β) for Alzheimer's Disease, Huntingtin forHuntington's Disease, α-synuclein for Parkinson's Disease, or α4integrin, CD25, or IL-23 for MS. In particular embodiments, the p97polypeptide is conjugated to an antibody or other agent thatspecifically binds to tumor necrosis factor (TNF-α, TNF-β) for thetreatment of other neurological conditions such as stroke, traumaticbrain injury (TBI), spinal stenosis, acute spinal cord injury, or spinalcord compression. In some embodiments, the p97 polypeptide is conjugatedto an interferon-β polypeptide or an antibody that specifically binds tothe alpha-subunit of the IL-2 receptor (CD25), α4 integrin, CD₂₀, CD52,IL-12, IL-23, the p40 subunit of IL-12 and IL-23, or at least one of theaxonal regrowth and remyelination inhibitors Nogo-A and LINGO-1, for thetreatment of MS. In specific embodiments, the p97 polypeptide isconjugated to daclizumab, natalizumab, rituximab, ocrelizumab,ofatumumab, alemtuzumab, or ustekinumab (CNTO 1275), for the treatmentof MS.

Also included are methods of treating pain in a subject in need thereof.General examples of pain include acute pain and chronic pain. In someinstances, the pain has at least one CNS component. Specific examples ofpain include nociceptive pain, neuropathic pain, breakthrough pain,incident pain, phantom pain, inflammatory pain including arthritic pain,or any combination thereof. In some aspects, the pain has acentrally-acting component, such as central pain syndrome (CPS), wherethe pain is associated with damage to or dysfunction of the CNS,including the brain, brainstem, and/or spinal cord.

In particular instances, the pain is nociceptive pain, optionallyvisceral, deep somatic, or superficial somatic pain. Nociceptive pain isusually caused by stimulation of peripheral nerve fibers that respond tostimuli approaching or exceeding harmful intensity (nociceptors), andmay be classified according to the mode of noxious stimulation; forexample, “thermal” (e.g., heat or cold), “mechanical” (e.g., crushing,tearing, cutting) and “chemical.” Visceral structures are highlysensitive to stretch, ischemia and inflammation, but relativelyinsensitive to other stimuli such as burning and cutting. Visceral painis most often diffuse, difficult to locate, and is sometimes referred toas having a distant, or superficial, structure. Visceral pain can beaccompanied by nausea and vomiting, and is sometimes described assickening, deep, squeezing, and dull. Deep somatic pain is usuallyinitiated by the stimulation of nociceptors in ligaments, tendons,bones, blood vessels, fasciae and muscles, and is often characterized asa dull, aching, or poorly localized pain. Examples include sprains andbroken bones. Superficial pain is mainly initiated by activation ofnociceptors in the skin or other superficial tissue, and is sharp,well-defined and clearly located. Examples of injuries that producesuperficial somatic pain include wounds and burns.

Neuropathic pain results from damage or disease affecting thesomatosensory system. It may be associated with abnormal sensationscalled dysesthesia, and pain produced by normally non-painful stimuli(allodynia). Neuropathic pain may have continuous and/or episodic(paroxysmal) components, the latter being compared to an electric shock.Common characteristics of neuropathic pain include burning or coldness,“pins and needles” sensations, numbness, and itching. Neuropathic painmay result from disorders of the peripheral nervous system or thecentral nervous system (e.g., brain, spinal cord). Neuropathic pain maybe characterized as peripheral neuropathic pain, central neuropathicpain, or mixed (peripheral and central) neuropathic pain.

Central neuropathic pain is found in spinal cord injury, multiplesclerosis, and strokes. Additional causes of neuropathic pain includediabetic neuropathy, herpes zoster infection, HIV-related neuropathies,nutritional deficiencies, toxins, remote manifestations of malignancies,immune mediated disorders, and physical trauma to a nerve trunk.Neuropathic pain also associates with cancer, mainly as a direct resultof a cancer or tumor on peripheral or central nerves (e.g., compressionby a tumor), or as a side effect of chemotherapy, radiation injury, orsurgery.

In some instances, the pain is breakthrough pain. Breakthrough pain ispain that comes on suddenly for short periods of time and is notalleviated by the subject's normal pain management regimen. It is commonin cancer patients who often have a background level of pain controlledby medications, but whose pain periodically “breaks through” themedication. Hence, in certain instances, the subject is taking painmedication, and is optionally a subject with cancer pain, e.g.,neuropathic cancer pain.

In certain instances, the pain is incident pain, a type of pain thatarises as a result of an activity. Examples include moving an arthriticor injured joint, and stretching a wound.

In specific instances, the pain is osteoarthritis, low back pain (orlumbago), including acute, sub-acute, and chronic low back pain (CLBP),bone cancer pain, or interstitial cystitis.

Osteoarthritis (OA), also referred to as degenerative arthritis ordegenerative joint disease or osteoarthrosis, is a group of mechanicalabnormalities involving degradation of joints, including articularcartilage and subchondral bone. Symptoms of OA may include joint pain,tenderness, stiffness, locking, and sometimes an effusion. OA may beinitiated by variety of causes, including hereditary, developmental,metabolic, and mechanical causes, most of which lead to the loss ofcartilage. When bone surfaces become less well protected by cartilage,bone may be exposed and damaged. As a result of decreased movementsecondary to pain, regional muscles may atrophy, and ligaments maybecome increasingly lax. Particular examples include osteoarthritis ofthe knee, and osteoarthritis of the hip.

Interstitial cystitis, or bladder pain syndrome, is a chronic,oftentimes severely debilitating disease of the urinary bladder. Ofunknown cause, it is characterized, for instance, by pain associatedwith the bladder, pain associated with urination (dysuria), urinaryfrequency (e.g., as often as every 10 minutes), urgency, and/or pressurein the bladder and/or pelvis.

In particular methods for treating pain, the p97 polypeptide isconjugated to an antibody or other agent that specifically binds to NGFor TrkA. In specific embodiments, the p97 polypeptide is conjugated totanezumab for the treatment of pain, optionally for the treatment ofosteoarthritis of the knee or hip, chronic low back pain, bone cancerpain, or interstitial cystitis.

Certain embodiments include combination therapies for treating pain. Forinstance, a subject with pain may be administered a p97-antibodyconjugate described herein, where the antibody specifically binds to atleast one pain-associated antigen, in combination with one or more painmedications, including analgesics and anesthetics. Exemplary analgesicsinclude, without limitation, paracetamol/acetaminophen; non-steroidalanti-inflammatory drugs (NSAIDS) such as salicylates (e.g., aspirin),propionic acid derivatives (e.g., ibuprofen, naproxen), acetic acidderivatives (e.g., indomethacin), enolic acid derivatives, fenamic acidderivatives, and selective COX-2 inhibitors; opiates/opioids andmorphinomimetics such as morphine, buprenorphine, codeine, oxycodone,oxymorphone, hydrocodone, dihydromorphine, dihydrocodeine, levorphanol,methadone, dextropropoxyphene, pentazocine, dextromoramide, meperidine(or pethidin), tramadol, noscapine, nalbuphine, pentacozine, papverine,papavereturn, alfentanil, fentanyl, remifentanil, sufentanil, andetorphine; and other agents, such as flupirtine, carbamazepine,gabapentin, and pregabalin, including any combination of the foregoing.

As noted above, certain subjects are about to undergo, are undergoing,or have undergone therapy with an otherwise cardiotoxic agent, that is,an agent that displays cardiotoxicity in its unconjugated form (an agentthat is not conjugated to p97). Such subjects can benefit fromadministration of a p97-agent conjugate, relative to administration ofthe agent alone, partly because p97 can exert a cardioprotective effecton otherwise cardiotoxic agents by a mechanism that is believed todiffer from its BBB transport properties. Hence, such subjects can betreated with a p97-cardiotoxic agent conjugate for a variety of diseaseconditions, including diseases of the CNS described herein, and diseasesrelating to peripheral, non-CNS tissues.

Exemplary cardiotoxic agents are described elsewhere herein, and can beidentified according to well-known in vivo diagnostic and in vitroscreening techniques. See Bovelli et al., 2010, supra; Inoue et al.,AATEX 14, Special Issue, 457-462, 2007; and Dorr et al., CancerResearch. 48:5222-5227, 1988.

For instance, subjects undergoing therapy with a suspected cardiotoxicagent can be monitored by imaging techniques to asses LV systolic anddiastolic dysfunction, heart valve disease, pericarditis and pericardialeffusion, and carotid artery lesions. LV fractional shortening and LVEFare the most common indexes of LV systolic function for cardiac functionassessment, for instance, during chemotherapy. Also, Doppler-deriveddiastolic indexes represent an early sign of LV dysfunction in patientsundergoing therapy, so that evaluation of mitral diastolic flow pattern,early peak flow velocity to atrial peak flow velocity (E/A) ratio,deceleration time of E wave and isovolumic relaxation time can be usefulto detect diastolic changes of LV function before systolic dysfunctionoccurs. Pulsed tissue Doppler may be performed during a standard Dopplerechocardiographic examination; it can be reliable in providingquantitative information on myocardial diastolic relaxation and systolicperformance (E′ wave, A′ wave and S wave velocity). Tissue Doppler of LVlateral mitral annulus has a recognized prognostic role and, incombination with PW Doppler of mitral inflow, provides accurateinformation about the degree of LV filling pressure. Early changes in LVmyocardial function have been identified by pulsed tissue Doppler ofmultiple LV sites, and can be relevant determinants of cardiotoxicity.

In particular embodiments, the cardiotoxic agent is a chemotherapeutic,and the subject has cancer. Specific examples of cancers include,without limitation, breast cancers, prostate cancers, gastrointestinalcancers, lung cancers, ovarian cancers, testicular cancers, head andneck cancers, stomach cancers, bladder cancers, pancreatic cancers,liver cancers, kidney cancers, squamous cell carcinomas, CNS or braincancers (described herein), melanomas, non-melanoma cancers, thyroidcancers, endometrial cancers, epithelial tumors, bone cancers, andhematopoietic cancers.

In specific embodiments, the subject has a Her2/neu-expressing cancer,such as a breast cancer, ovarian cancer, stomach cancer, aggressiveuterine cancer, or metastatic cancer, such as a metastatic CNS cancer,and the p97 polypeptide is conjugated to trastuzumab. Such patients canbenefit not only from the therapeutic synergism resulting from thecombination of p97 and trastuzumab, especially for CNS cancers, but alsofrom the reduced cardiotoxicity of trastuzumab, resulting from thepotential cardioprotective effects of p97.

As noted above, exemplary diseases that can be treated, ameliorated orprevented using the methods of the present invention include, but arenot limited to the following: various cancers, neurological conditions,conditions involving disturbances in iron metabolism,Mucopolysaccharidosis I (MPS I), MPS II, MPS IIIA, MPS IIIB,Metachromatic Leukodystropy (MLD), Krabbe, Pompe, CLN2, Tay-Sachs,Niemann-Pick A and B, and other lysosomal diseases. For each disease theconjugated agent would comprise a specific compound, protein or enzyme.For methods involving MPS I, the preferred compound or enzyme isα-L-iduronidase. For methods involving MPS II, the preferred compound orenzyme iduronate-2-sulfatase. For methods involving MPS IIIA, thepreferred compound or enzyme is heparan N-sulfatase. For methodsinvolving MPS IIIB, the preferred compound or enzyme isα-N-acetylglucosaminidase. For methods involving MetachromaticLeukodystropy (MLD), the preferred compound or enzyme is ArylsulfataseA. For methods involving Krabbe, the preferred compound or enzyme isGalactosylceramidase. For methods involving Pompe, the preferredcompound or enzyme is acid-alpha-glucosidase. For methods involving CLN,the preferred compound or enzyme is thioesterase. For methods involvingTay-Sachs, the preferred compound or enzyme is hexosaminidase A. Formethods involving Niemann-Pick A and B the preferred compound or enzymeis Acid Spingomyelinase. For methods involving other Glycogenosisdisorders the preferred compound or enzyme is glycolipidoses,mucopolysaccharidoses, oligosaccharidoses.

The p97 fragment-conjugates of the present invention can be administeredwith a “pharmaceutically acceptable carrier.” Such carriers encompassany of the standard pharmaceutical carriers, buffers and excipients,including phosphate-buffered saline solution, water, and emulsions (suchas an oil/water or water/oil emulsion), and various types of wettingagents and/or adjuvants. Suitable pharmaceutical carriers and theirformulations are described in Remington's Pharmaceutical Sciences (MackPublishing Co., Easton, 19th ed. 1995). Preferred pharmaceuticalcarriers depend upon the intended mode of administration of the activeagent. Typical modes of administration are described below.

The term “effective amount” means a dosage sufficient to produce adesired result on a health condition, pathology, disease of a subject orfor a diagnostic purpose. The desired result may comprise a subjectiveor objective improvement in the recipient of the dosage.

A “prophylactic treatment” is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of adisease, wherein treatment is administered for the purpose of decreasingthe risk of developing a pathology. The conjugate conjugates of theinvention may be given as a prophylactic treatment.

A “therapeutic treatment” is a treatment administered to a subject whoexhibits signs of pathology, wherein treatment is administered for thepurpose of diminishing or eliminating those pathological signs. Thesigns may be subjective or objective.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a p97 fragment-agent conjugate of thepresent invention and a pharmaceutically acceptable carrier.

The term “pharmaceutical composition” indicates a composition suitablefor pharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount ofthe p97 fragment-conjugate and a pharmaceutically acceptable carrier.

The conjugates may be administered by a variety of routes. For oralpreparations, the conjugates can be used alone or in combination withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents.

The p97 fragment-agent conjugates can be formulated into preparationsfor injection by dissolving, suspending or emulsifying them in anaqueous or nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The p97 fragment-agent conjugates can be utilized in aerosol formulationto be administered via inhalation. The conjugates of the presentinvention can be formulated into pressurized acceptable propellants suchas dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the p97 fragment-agent conjugates can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The conjugates of the present inventioncan be administered rectally via a suppository. The suppository caninclude vehicles such as cocoa butter, carbowaxes and polyethyleneglycols, which melt at body temperature, yet are solidified at roomtemperature.

Unit dosage forms of the p97 fragment-agent conjugates for oral orrectal administration as, for instance, syrups, elixirs, and suspensionsmay be provided wherein each dosage unit, for example, teaspoonful,tablespoonful, tablet or suppository, contains a predetermined amount ofthe composition containing active agent. Similarly, unit dosage formsfor injection or intravenous administration may comprise the conjugatein a composition as a solution in sterile water, normal saline oranother pharmaceutically acceptable carrier. The term “unit dosageform,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of conjugates of the present invention calculatedin an amount sufficient to produce the desired effect in associationwith a pharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular conjugate employed and the effect to beachieved, and the pharmacodynamics associated with each compound in thehost.

In practical use, the conjugates according to the invention can becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets, with the solid oral preparations being preferred over theliquid preparations.

With respect to transdermal routes of administration, methods fortransdermal administration of drugs are disclosed in Remington'sPharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds., MackPublishing Co., 1985). Dermal or skin patches are a preferred means fortransdermal delivery of the p97 fragment-agent conjugates of theinvention. Patches preferably provide an absorption enhancer such asDMSO to increase the absorption of the conjugates. Other methods fortransdermal drug delivery are disclosed in U.S. Pat. Nos. 5,962,012,6,261,595, and 6,261,595. Each of which is incorporated by reference inits entirety.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific agent, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven conjugate are readily determinable by those of skill in the art bya variety of means.

In each of these aspects, the compositions include, but are not limitedto, compositions suitable for oral, rectal, topical, parenteral(including subcutaneous, intramuscular, and intravenous), pulmonary(nasal or buccal inhalation), or nasal administration, although the mostsuitable route in any given case will depend in part on the nature andseverity of the conditions being treated and on the nature of the activeingredient. Exemplary routes of administration are the oral andintravenous routes. The compositions may be conveniently presented inunit dosage form and prepared by any of the methods well-known in theart of pharmacy.

In practical use, the conjugates according to the invention can becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets, with the solid oral preparations being preferred over theliquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. The percentageof an active agent in these compositions may, of course, be varied andmay conveniently be between about 2 percent to about 60 percent of theweight of the unit.

The conjugates of the invention are useful for therapeutic, prophylacticand diagnostic intervention in animals, and in particular in humans.

Compositions of the present invention may be administered encapsulatedin or attached to viral envelopes or vesicles. Liposomes are vesiclesformed from a bilayer membrane. Suitable vesicles include, but are notlimited to, unilamellar vesicles and multilamellar lipid vesicles orliposomes. Such vesicles and liposomes may be made from a wide range oflipid or phospholipid compounds, such as phosphatidylcholine,phosphatidic acid, phosphatidylserine, phosphatidylethanolamine,sphingomyelin, glycolipids, gangliosides, etc. using standardtechniques, such as those described in, e.g., U.S. Pat. No. 4,394,448.Such vesicles or liposomes may be used to administer conjugatesintracellularly and to deliver the conjugates to the target organs.Controlled release of a p97-composition of interest may also be achievedusing encapsulation (see, e.g., U.S. Pat. No. 5,186,941).

In certain aspects, the p97 polypeptide sequence and the agent are each,individually or as a pre-existing conjugate, bound to or encapsulatedwithin a particle, e.g., a nanoparticle, bead, lipid formulation, lipidparticle, or liposome, e.g., immunoliposome. For instance, in particularembodiments, the p97 polypeptide sequence is bound to the surface of aparticle, and the agent of interest is bound to the surface of theparticle and/or encapsulated within the particle. In some of these andrelated embodiments, the p97 polypeptide and the agent are covalently oroperatively linked to each other only via the particle itself (e.g.,nanoparticle, liposome), and are not covalently linked to each other inany other way; that is, they are bound individually to the sameparticle. In other embodiments, the p97 polypeptide and the agent arefirst covalently or non-covalently conjugated to each other, asdescribed herein (e.g., via a linker molecule), and are then bound to orencapsulated within a particle (e.g., immunoliposome, nanoparticle). Inspecific embodiments, the particle is a liposome, and the compositioncomprises one or more p97 polypeptides, one or more agents of interest,and a mixture of lipids to form a liposome (e.g., phospholipids, mixedlipid chains with surfactant properties). In some aspects, the p97polypeptide and the agent are individually mixed with the lipid/liposomemixture, such that the formation of liposome structures operativelylinks the p97 polypeptide and the agent without the need for covalentconjugation. In other aspects, the p97 polypeptide and the agent arefirst covalently or non-covalently conjugated to each other, asdescribed herein, and then mixed with lipids to form a liposome. The p97polypeptide, the agent, or the p97-agent conjugate may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980). The particle(s) or liposomes may further comprise othertherapeutic or diagnostic agents, such as cytotoxic agents.

Any route of administration which brings the conjugates into contactwith the target cells, tissue or organ may be used. The conjugates canbe administered peripherally or centrally. The conjugates may also beadministered intravenously or by intraperitoneally. The conjugates maybe administered locally or regionally.

The dosages to be administered will depend on individual needs andcharacteristics (age, weight, severity of condition, on the desiredeffect, the active agent used, and the chosen route of administrationand treatment regimen). Preferred dosages of p97 fragment-conjugatesrange from about 0.02 pmol/kg to about 2.5 nmol/kg, and particularlypreferred dosages range from 2-250 pmol/kg; alternatively, preferreddoses of the p97 fragment conjugate may be in the range of 0.02 to 2000mg/kg. These dosages will be influenced by the number of agent moietiesassociated with each p97 fragment molecule. In addition, dosages may becalculated based on the agent to be administered and the severity of thecondition to be treated. Empirical and theoretical methods fordetermining dose response relationships and optimizing the dosagesemployed an individual patients therapy are will known to one ofordinary skill in the art.

The p97 fragment-conjugates of the invention are, for example, usefulfor therapeutic and prophylactic intervention the treatment of lysosomalstorage diseases in animals, and in particular in humans. The subjectmethods find use in the treatment of a variety of different lysosomalstorage diseases. In certain embodiments, of particular interest is theuse of the subject methods in disease conditions where an active agenthaving desired activity has been previously identified, but in which theactive agent is not adequately targeted to the target site, area orcompartment. With such active agent, the subject methods can be used toenhance the therapeutic efficacy and therapeutic index of active agent.

The p97 fragment-conjugates of the invention are, for example, usefulfor delivering therapeutic or diagnostic agents across the blood brainbarrier.

Treatment is meant to encompass any beneficial outcome to a subjectassociated with administration of a conjugate including a reducedlikelihood of acquiring a disease, prevention of a disease, slowing,stopping or reversing, the progression of a disease or an ameliorationof the symptoms associated with the disease condition afflicting thehost, where amelioration or benefit is used in a broad sense to refer toat least a reduction in the severity of the disease or in a magnitude ofa parameter representative of the severity or presence of the disease,e.g., tissue damage, cell death, excess or harmful amounts of lysosomalstorage materials, symptoms, associated with the pathological conditionbeing treated, such as inflammation and pain associated therewith. Assuch, treatment also includes, but is not limited to, situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g., prevented from happening, or stopped,e.g., terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

A variety of hosts or subjects are treatable according to the subjectmethods. Generally such subjects are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys). In many embodiments, the hosts orsubjects will be humans.

Methods for identifying subjects with one or more of the diseases orconditions described herein are known in the art.

Also included are methods for imaging an organ or tissue component in asubject, comprising (a) administering to the subject a compositioncomprising a human p97 (melanotransferrin) polypeptide, or a variantthereof, where the p97 polypeptide is conjugated to a detectable entity,and (b) visualizing the detectable entity in the subject, organ, ortissue.

In particular embodiments, the organ or tissue compartment comprises thecentral nervous system (e.g., brain, brainstem, spinal cord). Inspecific embodiments, the organ or tissue compartment comprises thebrain or a portion thereof, for instance, the parenchyma of the brain.

A variety of methods can be employed to visualize the detectable entityin the subject, organ, or tissue. Exemplary non-invasive methods includeradiography, such as fluoroscopy and projectional radiographs,CT-scanning or CAT-scanning (computed tomography (CT) or computed axialtomography (CAT)), whether employing X-ray CT-scanning, positronemission tomography (PET), or single photon emission computed tomography(SPECT), and certain types of magnetic resonance imaging (MRI),especially those that utilize contrast agents, including combinationsthereof.

Merely by way of example, PET can be performed with positron-emittingcontrast agents or radioisotopes such as ¹⁸F, SPECT can be performedwith gamma-emitting contrast agents or radioisotopes such as ²⁰¹Tl,^(99m)Tc, ¹²³I, and ⁶⁷Ga, and MRI can be performed with contrast agentsor radioisotopes such as ³H, ¹³C, ¹⁹F, ¹⁷O, ²³Na, ³¹P, and ¹²⁹Xe, and Gd(gadolidinium; chelated organic Gd (III) complexes). Any one or more ofthese exemplary contrast agents or radioisotopes can be conjugated to orotherwise incorporated into a p97 polypeptide and administered to asubject for imaging purposes. For instance, p97 polypeptides can bedirectly labeled with one or more of these radioisotopes, or conjugatedto molecules (e.g., small molecules) that comprise one or more of theseradioisotopic contrast agents, or any others described herein.

EXAMPLES Example 1 Human p97 Digestion with Hydroxylamine

Even though previous studies have shown that soluble MTf is capable ofdelivering iron, paclitaxel and adriamycin across the BBB into thebrain, it was desired to determine if a smaller version of soluble MTfwas able to retain its ability to cross the BBB and function moreefficiently.

By analyzing the sequence of soluble MTf (see FIG. 1; residues 20-709 offull-length human MTf), it was determined that hydroxylamine, aninorganic compound, could shorten the soluble MTf sequence significantlywithout affecting its iron binding site. One of the resulting fragmentsof MTf is approximately 60-70 KDa in size (see FIG. 2). Completedigestion of soluble MTf with hydroxylamine was predicted to result infour fragments (˜60-70 KDa, ˜2.5 KDa, ˜5.5 KDa, and ˜5.8 KDa), the sizesof which are based on expected migration in a 1-D SDS-PAGE gel.Completely digested fragments of soluble MTf include amino acid residues1-564 (SEQ ID NO:1), residues 565-586 (SEQ ID NO:2), residues 587-637(SEQ ID NO:3), and residues 638-390 (SEQ ID NO:4).

Partially digested fragments are also predicted. For instance, partiallydigested fragments of soluble MTf include amino acid residues 1-586 (SEQID NO:5), residues 1-637 (SEQ ID NO:6), residues 565-637 (SEQ ID NO:7),residues 565-690 (SEQ ID NO:8), and residues 587-690 (SEQ ID NO:9).

The digestion was performed by dissolving 5 g hydroxylaminehydrochloride (sigma: 255580) in 5M NaOH. The pH was adjusted to 9.0.Human p97 was mixed with hydroxylamine solution, at a finalconcentration of 2.4M hydroxylamine. The mixture was incubated for 2-3days at 42° C. The reaction was terminated by adding 0.1 volume ofacetic acid or acidifying the mixture to PH 4.5 with glacial aceticacid. The mixture was cooled to 4° C. It was then dialyzed against 5%acetic acid O/N. Next it was dialyzed against PBS O/N.

Example 2 Bio-Distribution and Pharmacokinetics of the p97 60 kDFragment

In order to determine if the p97 fragment retained the ability of thefull length p97 to cross the BBB, the 60 kDa MTf fragment wasradiolabeled with ¹²⁵I and delivered into the mice through tail veininjection (FIG. 3). The bio-distribution and pharmacokinetics studiesdescribed herein show that MTf fragment was able to be absorbed rapidlyfrom the serum similar to MTf, while significant amount of IgG remainedin the circulation after the first 0.5 hour (FIG. 4).

The brain distribution of MTf, MTf fragment and IgG control was analyzedover 24 hour time period after a single I.V. injection. The data ispresented as the percentage of injected dose normalized to body mass (%ID/g BM; FIG. 5), as well as the ratio between radioactive counts (CPM)in one gram of tissue relative to one microliter of serum (Vd; FIG. 6).These results demonstrated that MTf fragment follow a similar profile asMTf with a gradual decrease over the 24 hour time period. When thedistribution of MTf, MTf fragment and IgG were normalized to the levelsin the serum (Vd), the results showed that both MTf and MTf fragmentdistributed to the brain 5× higher that of IgG. Taken together, thesedata suggest that MTf fragment is able to cross the BBB and accumulatein the brain similar to that of MTf. Similar analyses were performedstudying the heart, liver, kidney, lung and spleen, as shown in FIGS.9-18.

These results strongly suggest that MTf fragments have potential as analternative to full length or soluble MTf as a drug delivery carrier.One advantage to this would be to reduce the overall size of the carrierand the amount of protein per molecule of “therapeutic drug” that wouldhave to be delivered in each therapeutic dose.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. An isolated p97 polypeptide consisting ofan amino acid sequence selected from the group consisting of SEQ IDNO:1-8 and
 9. 2. A composition comprising a fragment of p97 thatconsists of (a) an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-8 and 9, (b)an amino acid sequence with atleast 97% identity along its lenght to a sequence of (a), or (c) anamino acid sequence that differs from a sequence of (a) by the additionor deletion of about 1,2,3,4, or 5 N-terminal and/or C-terminalresidues; and a therapeutic or diagnostic agent.
 3. The isolatedpolypeptide of claim 1 labeled with a label selected from the groupconsisting of fluorescent molecules, luminescent molecules, enzymes,substances having therapeutic activity, toxins, and radionuclides. 4.The isolated polypeptide of claim 1 conjugated to a therapeutic agent ordrug.
 5. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound comprising a p97 fragment covalently oroperatively linked to a therapeutic agent and a pharmaceuticallyacceptable excipient, wherein the p97 fragment consists of the aminoacid sequence set forth in SEQ ID NO:1-8 or
 9. 6. A conjugate,comprising a p97 polypeptide that consists of (a) an amino acid sequenceselected from the group consisting of SEQ ID No:1-8 and 9,(b) an aminoacid sequence with at least 97% identity along its length to a sequenceof (a), or (c) an amino acid sequence that differs from a sequence of(a) by the addition or deletion of about 1,2,3,4, or 5 N-terminal and/orC-terminal residues, where the p97 polypeptide is covalenty oroperatively linked to an agent, to form a p97-agent conjugate.
 7. Theconjugate of claim 6, where the agent is a small molecule, apolypeptide, or a label (detectable entity).
 8. The conjugate of claim7, where the small molecule is a cytotoxic or chemotherapeutic oranti-angiogenic agent selected from one or more of alkylating agents,anti-metabolites, anthracyclines, anti-tumor antiobiotics, platinums,type I topoisomerase inhibitors, type II topoisomerase inhibitors, vincaalkaloids, and taxanes.
 9. The conjugate of claim 7, where the smallmolecule is selected from one or more of chlorambucil, cyclophosphamide,cilengitide, lomustine (CCNU), melphalan, procarbazine, thiotepa,carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin,gefitinib, erlotinib idarubicin, temozolomide, epirubicin, mitoxantrone,bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecins,irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate,teniposide, temsirolimus, everolimus, vincristine, vinblastine,vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib,sorafenib, pazopanib, sunitnib, vatalanib, geftinib, erlotinib, AEE-788,dichoroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepizil,galantamine, memantine, rivastigmine, tacrine, rasigiline, naltrexone,lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant,isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimeracetate, fingolimod, and mitoxantrone, including pharmaceuticallyacceptable salts and acids thereof.
 10. The conjugate of claim 7, wherethe polypeptide is an antibody or antigen-binding fragment thereof. 11.The conjugate of claim 10, wherein the antibody or antigen-bindingfragment thereof specifically binds to one or more of human Her2/neu,Her1/EGFR, CD20, VEGF, CD52, CD33, CTLA-4, tenascin, alpha-4 (α4)integrin, IL-12, IL-23, the p40 subunit of IL-12/IL-23, amyloid-β(Aβ),Huntingtin, CD25, nerve growth factor (NGF), TrkA, TNF-α, TNF-β, orα-synuclein.
 12. The conjugate of claim 10, where the antibody isselected from one or more of trastuzumab, cetuximab, daclizumab,tanezumab, 3F8, abagovomab, adalimumab, adecatumumab, afutuzumab,alemtuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab,bectumomab, belimumab, bevacizumab, bivatuzumab (mertansine),brentuximab vedotin, cantuzumab (mertansine), cantuzumab (ravtansine),capromab (pendetide), catumaxomab, certolizumab, citatuzumab (bogatox),cixutumumab, clivatuzumab (tetraxetan), conatumumab, dacetuzumab,dalotuzumab, detumomab, drozitumab, ecromeximab, edrecolomab,elotuzumab, enavatuzumab, ensituximab, epratuzumab, ertumaxomab,etaracizumab, farletuzumab, FBTA05, figitumumab, flanvotumab, galiximab,gemtuzumab, ganitumab, gemtuzumab (ozogamicin), girentuximab,glembatumumab (vedotin), golimumab, ibritumomab tiuxetan, icrucumab,igovomab, indatuximab ravtansine, infliximab, intetumumab, inotuzumabozogamicin, ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab,lintuzumab, lorvotuzumab (mertansine), lucatumumab, lumiliximab,mapatumumab, matuzumab, milatuzumab, mitumomab, mogamulizumab,moxetumomab (pasudotox), nacolomab (tafenatox), naptumomab(estafenatox), narnatumab, necitumumab, nimotuzumab, nivolumab,Neuradiab® (with or without radioactive iodine), NR-LU-10, ofatumumab,olaratumab, onartuzumab, oportuzumab (monatox), oregovomab, panitumumab,patritumab, pemtumomab, pertuzumab, pritumumab, racotumomab, radretumab,ramucirumab, rilotumumab, rituximab, robatumumab, samalizumab,sibrotuzumab, siltuximab, tabalumab, taplitumomab (paptox), tenatumomab,teprotumumab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650,tositumomab, TRBS07, tucotuzumab (celmoleukin), ublituximab, urelumab,veltuzumab, volociximab, votumumab, and zalutumumab, includingantigen-binding fragments thereof.
 13. The conjugate of claim 7, wherethe polypeptide is an interferon-βpolypeptide, or an active fragment orvariant thereof.
 14. The conjugate of claim 7, where the polypeptideassociates with a lysosomal storage disease.
 15. The conjugate of claim14, where the polypeptide is selected from one or more ofaspartylglucosaminidase, acid lipase, cysteine transporter, Lamp-2,α-galactosidase A, acid ceramidase, α-L-fucosidase,β-hexosaminidase A,GM2-ganglioside activator (GM2A), αD-mannosidase, β-D-mannosidase,arylsulfatase A, saposin B, neuraminidase, α-N acetylglucosaminidasephosphotransferase, phosphotransferase Υ-subunit, L-iduronidase,iduronate-2-sulfatase, heparan-N-sulfatase, α-N-acetylglucosaminidase,acetylCoA:N acetyltransferase, N-acetylglucosamine 6-sulfatase,galactose 6-sulfatase,β-galactosidase, N-acetylgalactosamine4-sulfatase, hyaluronoglucosaminidase, sulfatases, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsinA, cathepsin K, α-galactosidase B, NPC1, NPC2, sialin, and sialic acidtransporter, including active fragments and variants thereof.
 16. Theconjugate of claim 7, where the detectable entity is selected from oneor more of diatrizoic acid, a radioisotope, a fluorophore/fluorescentdye, and a nanoparticle.
 17. The conjugate of claim 7, where the agentis a cardiotoxic agent.
 18. The conjugate of claim 17, where thecardiotoxic agent is an anthracycline/anthraquinolone, cyclophosphamide,antimetabolite, antimicrotubule agent, tyrosine kinase inhibitor,bevacizumab, or trastuzumab.
 19. The conjugate of claim 17, where thecardiotoxic agent is cyclopentenyl cytosine, 5-fluorouracil,capecitabine, paclitaxel, docataxel, adriamycin, doxorubucin,epirubicin, emetine, isotamide, mitomycin C, erlotinib, gefitinib,imatinib, sorafenib, sunitinib, cisplatin, thalidomide, busulfan,vinblastine, bleomycin, vincristine, arsenic trioxide, methotrexate,rosiglitazone, or mitoxantrone.
 20. A composition, comprising aconjugate of claim 6, and a pharmaceutically acceptable carrier.
 21. Theconjugate of claim 7, where the polypeptide is etanercept, or an activefragment or variant thereof.